WO2000045771A2 - Suppression de la transformation de cellules par le facteur de transcription egr - Google Patents

Suppression de la transformation de cellules par le facteur de transcription egr Download PDF

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WO2000045771A2
WO2000045771A2 PCT/US2000/002799 US0002799W WO0045771A2 WO 2000045771 A2 WO2000045771 A2 WO 2000045771A2 US 0002799 W US0002799 W US 0002799W WO 0045771 A2 WO0045771 A2 WO 0045771A2
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egr
expression
cell
cells
nucleic acid
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WO2000045771A3 (fr
WO2000045771A9 (fr
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Daniel Mercola
Eileen Adamson
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Daniel Mercola
Eileen Adamson
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Priority to AU44487/00A priority Critical patent/AU4448700A/en
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Publication of WO2000045771A3 publication Critical patent/WO2000045771A3/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention generally relates generally to the fields of molecular biology and cancer biology and treatment.
  • EGR-1 is a nuclear phosphoprotein that comprises three zinc finger motifs in the C- terminal portion of the molecule that confers specific DNA-binding properties to the molecule.
  • a growing body of circumstantial evidence indicates that EGR-1 may have functional effects in regulating cell growth, differentiation and development.
  • An approach to understanding the underlying molecular basis for these EGR-1 functions has been to study signal transduction events associated with the expression of EGR-1 and the possible modulation of expression of TGF- ⁇ l.
  • TGF- ⁇ l belongs to the TGF superfamily of cytokines that have been implicated in the regulation of growth, differentiation, development, and apoptosis. TGF- ⁇ l may play?a fole in modulating the activity of PAI- 1 and fibronectin (FN).
  • PAI- 1 is thought to participate in the stabilization of the extracellular matrix by either interacting with structures containing a urokinase plasminogen activator.
  • PAI-1 is also thought to inhibit the serine protease activity of a urokinase plasminogen activator.
  • FN plays a role in anchoring cells to the extracellular matrix. Inhibition of FN expression leads to a loss of FN from the cell surface and may be related to oncogenic transformation in vitro and may be related to tumorigenic and metastatic phenotypes in vivo. Although these various cellular components are known, their complex interactions generally, and particularly how they relate to tumor cells, has not been appreciated.
  • This invention satisfies this need and provides related advantages as well by providing methods of regulating the growth of cancer cells by promoting or supplementing the expression of EGR, or fragments thereof, in cells.
  • FIG. 1A depicts a Western blot analysis of EGR-1 expression in a series of EGR-1 stably transfected H4 cells.
  • FIG. IB depicts the presence of radiolabled PAI-1 produced by a series of EGR- 1 stably transfected H4 cells in the presence or absence of rhTGF- ⁇ 1.
  • FIG. 1G depicts densiometic analysis of PAI-1 expression in the absence (white bars) and presence (solid bars) of rhTGF- ⁇ 1. The insert shows the correlation of EGR-1 expression and PAI-1 expression.
  • FIG. 2A depicts the presence of radiolabled FN produced by a series of EGR-1 stably transfected H4 cells in the presence or absence of rhTGF- ⁇ 1.
  • FIG. IB depicts densiometic analysis of FN expression in the absence (white bars) and presence (solid bars) of rhTGF- ⁇ 1. The insert shows the correlation of EGR-1 expression and FN expression.
  • FIG. 3 A depicts the secretion of PAI-1 and FN in H4 cells in response to differing concentrations of rhTGF- ⁇ 1.
  • FIG.3B depicts densiometric analysis of the relative expression levels of PAI-1 and FN in response to differing concentrations of rhTGF- ⁇ 1.
  • FIG. 4 depicts the effect of anti-TGF- ⁇ 1>2j3 antibody on the secretion of PAI-Tan ⁇ * FN in a series of EGR-1 stably transfected H4 cells.
  • FIG. 5 depicts the effect of an application of antisense TGF- ⁇ l oligonucleotides on the secretion of PAI-1 and FN in a series of EGR-1 stably transfected H4 cells.
  • FIG. 6A depicts a schematic representation of the human FN promoter with the location of two GCE sites and transcription start sites indicated as shaded boxes.
  • FIG. 6B depicts gel- shift analysis of FLAG-tagged-EGR-1 (wild type) or mutant FLAG-tagged-EGR- 1 ⁇ S348A/A340S to detect binding with Probe A or Probe B in the presence or absence of anti- ERG-1 antibody or anti-Spl antibody.
  • the protein-DNA complexes are indicated by the arrow.
  • FIG. 6C depicts gel-shift analysis of GST-EGR-1 fusion protein to detect binding with Probe A or Probe B in the presence or absence of competing GCE sequences
  • the protein-DNA complexes are indicated by the arrow.
  • FIG. 6D depicts a gel-shift assay using nuclear protein extracts from a series of EGR-1 stably transfected H4 cells, Probe A or Probe B, in the presence or absence of anti-Spl antibody, anti-EGR-1 antibody or competing GCE sequences. DNA-protein complexes are indicated by the arrow.
  • FIG. 7A depicts the attachment of a series of EGR-1 stably transfected H4 cells to untreated polystyrene tissue culture dishes or Petri dishes over time.
  • FIG. 7B depicts the attachment of a series of EGR-1 stably transfected H4 cells to standard 96-well polystyrene ELISA plates pretreated with FN or PAI- 1 over time.
  • FIG. 8 depicts the attachment of a series of EGR-1 stably transfected H4 cells to standard 96-well polystyrene ELISA plates pretreated with PAI-1 in the presence or absence of anti-API-1 antibody or the peptides GRGDSP or GRGESP.
  • FIG. 9A depicts the attachment of a series of EGR-1 stably transfected H4 cells to standard 96-well ELISA plates pretreated with 5 ⁇ g ml of FN in the presence or absence of anti- PAI-1 antibody or the peptides GRGDSP or GRGESP.
  • FIG. 9B depicts the attachment of a series of EGR-1 stably transfected H4 cells to standard 96-well ELISA plates pretreated with 0.25 ⁇ g/ml of FN in the presence or absence of anti-PAI-1 antibody.
  • FIG. 10 depicts a proposed model for the mechanism of the suppression of transformation of cells by EGR- 1.
  • EGR- 1 which the inventors expressly do not wisRTo be bound to, but provide for clarification, the expression of FN receptor is regulated by FN and TGF- ⁇ l .
  • EGR-1 transactivates both TGF- ⁇ l and FN genes leading to increased steady state synthesis and secretion of TGF- ⁇ l and FN.
  • Secreted TGF- ⁇ l becomes activated and induces increased steady state PAI-1 expression via TGF- ⁇ l receptor-mediated signal transduction.
  • Both FN and PAI-1 augment ECM formation and density-dependent growth control.
  • FIG. 11 presents a schematic diagram of the truncated forms of Egr-1 and their calculated molecular weights. Fragments were constructed as described in U.S. Patent No. 5,837,692 to Mercola et al., issued November 17, 1998, using standard recombinant DNA techniques. The truncated forms contain the thirteen initial amino acids from the first methionine to proline, in order to initiate translation. The fragments were cloned into pGEM-1 (Promega) to effect their synthesis in vitro, and into eukaryotic vectors driven by RSV for expression in eukaryotic cells such as NTH3T3 cells. The four principal domains of Egr-1 are designated A, B, C and D. The DNA-binding zinc-finger domain is indicated (domain C).
  • the present invention recognizes that the expression of EGR can decrease metastasis of tumor cells.
  • the methods of the present invention involve transfecting a cell with a nucleic acid molecule encoding a mammalian EGR or a fragment thereof comprising the zinc finger domain of an EGR-1 or a nucleic acid sequence that hybridizes to any of the appropriate EGR-1 nucleic acid sequences described herein under standard hybridization conditions and that encodes a polypeptide that increases the expression of an anti-metastatic factor in a cell or interferes with the metastasis of a tumor cell.
  • the present invention also includes methods for identifying compounds or compositions that increase the expression of EGR-1 in a cell and compounds or compositions, including pharmaceutical compositions, that are identified by these methods.
  • a first aspect of the present invention is a method for increasing expression of an anti- metastatic factor in a tumor cell including administering to a tumor cell a vector comprising a nucleic acid sequence encoding EGR or a nucleic acid sequence that encodes an active portion of
  • a second aspect of the present invention is a method for interfering with the metastasis of a tumor cell including administering to a tumor cell a vector comprising a nucleic acid sequence encoding EGR or a nucleic acid sequence that encodes an active portion of EGR.
  • a third aspect of the present invention is a method for identifying compounds or compositions that increase the expression of EGR in a cell.
  • a fourth aspect of the present invention is a method for identifying compounds or compositions that increase or decrease the expression of EGR in a cell by contacting a cell with a test compound and measuring the expression of EGR in said cell and compositions, including pharmaceutical compositions, identified by this method.
  • a fifth aspect of the present invention is a method for interfering with proliferation of a tumor cell in a mammal by administering to a mammal a vector comprising a nucleic acid sequence encoding an EGR or a nucleic acid sequence that encodes an active portion of an EGR, wherein said tumor cell exhibits increased expression of at least one anti-metastatic factor upon said administering.
  • isolated polynucleotide refers to a polynucleotide of genomic, cDNA, or synthetic origin, or some combination thereof, which by virtue of its origin, the isolated polynucleotide (1) is not associated with the cell in which the isolated polynucleotide is found in nature, or (2) is operably linked to a polynucleotide that it is not linked to in nature.
  • the isolated polynucleotide can optionally be linked to promoters, enhancers, or other regulatory sequences.
  • isolated protein refers to a protein of cDNA, recombinant RNA, or synthetic origin, or some combination thereof, which by virtue of its origin the isolated protein (1) is not associated with proteins normally found within nature, or (2) is isolated from the cell in which it normally occurs, or (3) is isolated free of other proteins from the same cellular source, for example, free of cellular proteins), or (4) is expressed by a cell from a different species, or (5) does not occur in nature.
  • Polypeptide is used herein as a generic term to refer to native protein, fragments, or analogs of a polypeptide sequence.
  • Active fragment refers to a fragment of a parent molecule, such as an organic molecule, nucleic acid molecule, or protein or polypeptide, or combinations thereof, that retains at least one activity of the parent molecule.
  • Naturally occurring refers to the fact that an object can be found in nature.
  • a polypeptide or polynucleotide sequence that is present in an organism, including viruses, that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally occurring.
  • Operaably linked refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner.
  • a control sequence operably linked to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.
  • Control sequences refer to polynucleotide sequences that effect the expression of coding and non-coding sequences to which they are ligated. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal biding site, and transcription termination sequences; in eukaryotes, generally, such control sequences include promoters and transcription termination sequences.
  • the term control sequences is intended to include components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.
  • Polynucleotide refers to a polymeric form of nucleotides of a least ten bases in length, either ribonucleotides or deoxynucleotides or a modified from of either type of nucleotide.
  • the term includes single and double stranded forms of DNA or RNA.
  • Directly in the context of a biological process or processes, refers to direct causation of a process that does not require intermediate steps, usually caused by one molecule contacting or binding to another molecule (the same type or different type of molecule). For example, molecule A contacts molecule B, which causes molecule B to exert effect X that is part of a biological process.
  • Sequence homology refers to the proportion of base matches between two nucleic acid sequences or the proportion of amino acid matches between two amino acid sequences. When sequence homology is expressed as a percentage, for example 50%, the percentage denotes the proportion of matches of the length of sequences from a desired sequence that is compared to some other sequence. Gaps (in either of the two sequences) are permitted to maximize matching; gap lengths of 15 bases or less are usually used, 6 bases or less are preferred with 2 bases or less more preferred.
  • the sequence homology between the target nucleic acid and the oligonucleotide sequence is generally not less than 17 target base matches out of 20 possible oligonucleotide base pair matches (85%); preferably not less than 9 matches out of 10 possible base pair matches (90%), and most preferably not less than 19 matches out of 20 possible base pair matches (95%).
  • “Selectively hybridize” refers to detectably and specifically bind. Polynucleotides, oligonucleotides and fragments thereof selectively hybridize to target nucleic acid strands, under hybridization and wash conditions that minimize appreciable amounts of detectable binding to nonspecific nucleic acids.
  • High stringency conditions can be used to achieve selective hybridization conditions as known in the art.
  • the nucleic acid sequence homology between the polynucleotides, oligonucleotides, and fragments thereof and a nucleic acid sequence of interest will be at least 30%, and more typically and preferably of at least 40%, 50%, 60%, 70%, 80% or 90%.
  • Hybridization and washing conditions are typically performed at high stringency according to conventional hybridization procedures. Positive clones are isolated and sequenced. For example, a full length polynucleotide sequence can be labeled and used as a hybridization probe to isolate genomic clones from an appropriate target library as they are known in the art.
  • Typical hybridization conditions and methods for screening plaque lifts and other purposes are known in the art (Benton and Davis, Science 196: 180 (1978); Sambrook et al., supra, (1989)),
  • Two amino acid sequences are homologous if there is a partial or complete identity between their sequences. For example, 85% homology means that 85% of the amino acids are identical when the two sequences are aligned for maximum matching. Gaps (in either of the two sequences being matched) are allowed in maximizing matching; gap lengths of 5 or less are preferred with 2 or less being more preferred.
  • two protein sequences are homologous, as this term is used herein, if they have an alignment score of at least 5 (in standard deviation units) using the program ALIGN with the mutation data matrix and a gap penalty of 6 or greater (Dayhoff, in Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, volume 5, pp. 101-110 (1972) and Supplement 2, pp. 1-10).
  • the two sequences or parts thereof are more preferably homologous if their amino acids are greater than or equal to 30% identical when optimally aligned using the ALIGN program.
  • “Corresponds to” refers to a polynucleotide sequence is homologous (for example is identical, not strictly evolutionarily related) to all or a portion of a reference polynucleotide sequence, or that a polypeptide sequence is identical to all or a portion of a reference polypeptide sequence.
  • the term “complementary to” is u sed herein to mean that the complementary sequence is homologous to all or a portion of a reference polynucleotide sequence.
  • the nucleotide sequence TAT AC corresponds to a reference sequence
  • TAT AC is complementary to a reference sequence GTATA.
  • a reference sequence is a defined sequence used as a basis for a sequence comparison; a reference sequence can be a subset of a larger sequence, for example, as a segment of a full length cDNA or gene sequence given in a sequence listing, or may comprise a complete cDNA or gene sequence. Generally, a reference sequence is at least 20 nucleotides in length, frequently at least 25 nucleotides in length, and often at least 50 nucleotides in length.
  • two polynucleotides can each (1) comprise a sequence (for example a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) may further comprise a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity.
  • a comparison widow refers to a conceptual segment of at least 20 contiguous nucleotide positions wherein a polynucleotide sequence may be compared to a reference sequence of at least 20 contiguous nucleotides and wherein the portion of the polynucleotide sequence in the comparison window can comprise additions and deletions (for example, gaps) of 20 percent or less as compared to the reference sequence (which would not comprise additions or deletions) for optimal alignment of the two sequences.
  • Optimal alignment of sequences for aligning a comparison window can be conducted by the local homology algorithm (Smith and Waterman, Adv. Appl. Math., 2:482 (1981)), by the homology alignment algorithm (Needleman and Wunsch, J. Mol.
  • Sequence identity means that two polynucleotide sequences are identical (for example, on a nucleotide-by-nucleotide basis) over the window of comparison.
  • Percentage of sequence identity is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (for example, the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • Substantial identity denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 30 percent sequence identity, preferably at least 50 to 60 percent sequence, more usually at least 60 percent sequence identity as compared to a reference sequence over a comparison window of at least 20 nucleotide positions, frequently over a window of at least 25 to 50 nucleotides, wherein the percentage of sequence identity is calculated by comparing the reference sequence to the polynucleotide sequence that may include deletions or addition which total 20 percent or less of the reference sequence over the window of comparison.
  • Substantial identity as applied to polypeptides herein means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 30 percent sequence identity, preferably at least 40 percent sequence identity, and more preferably at least 50 percent sequence identity, and most preferably at lest 60 percent sequence identity. Preferably, residue positions, which are not identical, differ by conservative amino acid substitutions.
  • Constant amino acid substitutions refer to the interchangeability of residues having similar side chains.
  • a group of amino acids having aliphatic side chains is glycine, alanine, vaiine, leucine, and isoleucine
  • a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine
  • a group of amino acids having amide-containing side chains is
  • Preferred conservative amino acid substitution groups are: valine-leucine-isoleucine; phenylalanine-tyrosine; lysine-arginine; alanine-valine; glutamic-aspartic; and asparagine- glutamine.
  • Modulation refers to the capacity to either enhance or inhibit a functional property of a biological activity or process, for example, enzyme activity or receptor binding. Such enhancement or inhibition may be contingent on the occurrence of a specific event, such as activation of a signal transduction pathway and/or may be manifest only in particular cell types.
  • Module refers to a chemical (naturally occurring or non-naturally occurring), such as a biological macromolecule (for example, nucleic acid, protein, non-peptide or organic molecule) or an extract made from biological materials, such as prokaryotes, bacteria, eukaryotes, plants, fungi, multicellular organisms or animals, invertebrates, vertebrates, mammals and humans, including, where appropriate, extracts of: whole organisms or portions of organisms, cells, organs, tissues, fluids, whole cultures or portions of cultures, or environmental samples or portions thereof.
  • a biological macromolecule for example, nucleic acid, protein, non-peptide or organic molecule
  • an extract made from biological materials such as prokaryotes, bacteria, eukaryotes, plants, fungi, multicellular organisms or animals, invertebrates, vertebrates, mammals and humans, including, where appropriate, extracts of: whole organisms or portions of organisms, cells, organs, tissues, fluids, whole cultures or portions of cultures, or environmental samples
  • Modulators are typically evaluated for potential activity as inhibitors or activators (directly or indirectly) of a biological process or processes (for example, agonist, partial antagonist, partial agonist, antagonist, antineoplastic, cytotoxic, inhibitors of neoplastic transformation or cell proliferation, cell proliferation promoting agents, antiviral agents, antimicrobial agents, antibacterial agents, antibiotics, and the like) by inclusion in assays .described herein.
  • the activity of a modulator may be known, unknown or partially known.
  • Test compound refers to a chemical, compound or extract to be tested by at least one method of the present invention to be a putative modulator.
  • a test compound can be of any chemical nature, including but not limited to small molecules such as drugs, proteins or antibodies or active fragments thereof, nucleic acids such as DNA or RNA that can be provided in a vector such as a viral vector, antisense nucleic acid molecules and ribozymes.
  • a test compound is usually not known to bind to the target of interest, but that not need be the case.
  • Cons ⁇ ol test compound refers to a compound known to bind to the target (for example, a known agonist, antagonist, partial agonist or inverse agonist).
  • Test compound does not typically include a chemical added to a mixture as a control condition that alters the function of the target to determine signal specificity in an assay.
  • control chemicals or conditions include chemicals that (1) non-specifically or substantially disrupt protein structure (for example denaturing agents such as urea or guandium, sulfhydryl reagents such as dithiotritol and beta-mercaptoethanol), (2) generally inhibit cell metabolism (for example mitochondrial uncouples) and (3) non-specifically disrupt electrostatic or hydrophobic interactions of a protein (for example, high salt concentrations or detergents at concentrations sufficient to non-specifically disrupt hydrophobic or electrostatic interactions).
  • non-specifically or substantially disrupt protein structure for example denaturing agents such as urea or guandium, sulfhydryl reagents such as dithiotritol and beta-mercaptoethanol
  • cell metabolism for example mitochondrial uncouples
  • non-specifically disrupt electrostatic or hydrophobic interactions of a protein for example, high salt concentrations or detergent
  • test compound also does not typically include ⁇ e chemicals known to be unsuitable for a therapeutic use for a particular indication due to toxicity of the subject.
  • various predetermined concentrations of test compound are used for determining their activity. If the molecular weight of a test compound is known, the following ranges of concentrations can be used: between about 0.001 micromolar and about 10 millimolar, preferably between about 0.01 micromolar and about 1 millimolar, more preferably between about 0.1 micromolar and about 100 micromolar.
  • concentration of test compound used can be expressed on a weight to volume basis.
  • the following ranges of concentrations can be used: between about 0.001 micrograms/ml and about 1 milligram/ml, preferably between about 0.01 micrograms/ml and about 100 micrograms/ml, and more preferably between about 0.1 micrograms/ml and about 10 micrograms/ml.
  • Target refers to a biochemical entity involved in a biological process. Targets are typically proteins that play a useful role in the physiology or biology of an organism. A therapeutic chemical typically binds to a target to alter or modulate its function. As used herein, targets can include, but not be limited to, cell surface receptors, G-proteins, G-protein coupled receptors, kinases, phosphatases, ion channels, Upases, phosholipases, nuclear receptors, intracellular structures, tubules, tubulin, and the like.
  • Label' or labeld refers to incorporation of a detectable marker, for example by incorporation of a radiolabled compound or attachment to a polypeptide of moieties such as biotin that can be detected by the binding of a section moiety, such as marked avidin.
  • a detectable marker for example by incorporation of a radiolabled compound or attachment to a polypeptide of moieties such as biotin that can be detected by the binding of a section moiety, such as marked avidin.
  • Various methods of labeling polypeptide, nucleic acids, carbohydrates, and other biological or organic molecules are known in the art.
  • Such labels can have a variety of readouts, such as radioactivity, fluorescence, color, chemiluminescence or other readouts known in the art or later developed.
  • the readouts can be based on enzymatic activity, such as beta-galactosidase, beta-lactamase, horseradish peroxidase, alkaline phosphatase, luciferase; radioisotopes such as 3 H, , C, 35 S, 125 I or 131 I); fluorescent proteins, such as green fluorescent proteins; or other fluorescent labels, such as FITC, rhodamine, and lanthanides. Where appropriate, these labels can be the product of the expression of reporter genes, as that term is understood in the art. Examples of reporter genes are beta-lactamase (U.S. Patent No. 5,741,657 to Tsien et al., issued April 21, 1998) and green fluorescent protein (U.S. Patent No. 5,777,079 to Tsien et al.., issued July 7, 1998; U.S. Patent No. 5,804,387 to Cormack et al., issued September 8, 1998).
  • enzymatic activity such as beta-
  • substantially pure refers to an object species or activity that is the predominant species or activity present (for example on a molar basis it is more abundant than any other individual species or activities in the composition) and preferably a substantially purified f action is a composition wherein the object species or activity comprises at least about 50 percent (on a molar, weight or activity basis) of all macromolecules or activities present.
  • object species or activity comprises at least about 50 percent (on a molar, weight or activity basis) of all macromolecules or activities present.
  • as substantially pure composition will comprise more than about 80 percent of all macromolecular species or activities present in a composition, more preferably more than about 85%,_90%, 95% and 99%.
  • the object species or activity is purified to essential homogeneity, wherein contaminant species or activities cannot be detected by conventional detection methods) wherein the composition consists essentially of a single macromolecular species or activity.
  • an activity may be caused, directly or indirectly, by a single species or a plurality of species within a composition, particularly with extracts.
  • “Pharmaceutical agent or drug” refers to a chemical, composition or activity capable of inducing a desired therapeutic effect when property administered by an appropriate dose, regime, route of administration, time and delivery modality.
  • “Pharmaceutically effective amount” refers to an appropriate dose, regime, route of administration, time and delivery modality associated with the delivery of an amount of a compound or composition to cause a desired effect. Such pharmaceutically effective amount can be determined using methods described herein or by the United States Food and Drug Administration (USFDA).
  • USFDA United States Food and Drug Administration
  • nucleic acid molecule "encodes" a polypeptide if transcription of the nucleic acid molecule and translation of the mRNA produce the polypeptide.
  • nucleic acid molecules of the present invention include those whose nucleotide sequence encodes a polypeptide directly, such as cDNA, or whose nucleotide sequence includes introns that are spliced out upon transcription into mRNA, such as genomic DNA. It also includes nucleic acid molecules having sequences which are degenerate versions of any of the aforementioned nucleotide sequences.
  • the present invention recognizes that the expression of EGR can decrease metastasis of tumor cells and increase the expression of anti-metastatic factors.
  • the present invention includes several general and useful aspects, including: 1) a method for increasing expression of an anti-metastatic factor in a tumor cell including administering to a tumor cell a vector comprising a nucleic acid sequence encoding EGR or a nucleic acid sequence that encodes an active portion of EGR;
  • a method for interfering with the metastasis of a tumor cell including administering to a tumor cell a vector comprising a nucleic acid sequence encoding EGR or a nucleic acid sequence that encodes an active portion of EGK;
  • compositions including pharmaceutical compositions, identified by 3);
  • I A method for increasing expression of an anti-metastatic factor in a tumor cell, interfering with the metastasis of a tumor cell, and inhibiting the growth of a tumor cell.
  • this method is used to increase the expression of at least one anti-metastatic factor in a tumor cell or interferes with the metastasis of a tumor cell.
  • This method can also be used to inhibit the growth of a tumor cell.
  • Tumors whose growth can be inhibited by the expression of mammalian EGR include cancer cells derived from mesoderm, ectoderm or endoder , such as fibrosarcoma, breast carcinoma, glioblastoma, osteogenic sarcoma, glioblastoma, myelodysplastic syndrome, small cell lung carcinoma, non- small cell lung carcinoma, leukemia, acute myelogenous leukemia melanoma and prostate carcinoma.
  • the cell is a transformed culture cell such as from fibrosarcoma, breast carcinoma, glioblastoma, osteogenic sarcoma, glioblastoma, myelodysplastic syndrome, small cell lung carcinoma, non-small cell lung carcinoma, leukemia, acute myelogenous leukemia, melanoma and prostate carcinoma.
  • “Mammalian EGR” or “EGR” refers to a polypeptide of the EGR gene family having activity as a transcription factor. This includes the polypeptides encoded by the mouse Egr-1 gene, the human EGR-1 gene, the human EGR2 gene, the human EGR3 gene, the human EGR4 gene and other mammalian EGR genes identifiable as follows.
  • a mammalian EGR is characterized by having 30% overall amino acid sequence identity with at least one or the foregoing mammalian EGRs. It has a zinc finger domain with at least 80% amino acid identity with the zinc finger domain of at least one of the foregoing mammalian EGRs.
  • a mammalian EGR is also characterized by its ability to bind with at least one GCE site.
  • the DNA and amino acid sequence of mouse Egr-1 is given in U.S. Patent No. 5,837,692 to Mercola et al., issued November 11, 1998.
  • the DNA and deduced amino acid sequences of human EGRl is given in U.S. Patent No. 5,837,692 to Mercola et al., issued November 11, 1998.
  • the DNA and deduced amino acid sequence of human EGR2 is given in U.S. Patent No. 5,837,692 to Mercola et al., issued November 11, 1998.
  • the DNA and deduced amino acid sequence of human EGR3 is given in U.S. Patent No. 5,837,692 to Mercola et al., issued November 11, 1998.
  • a partial DNA and amino acid sequence of human EGR4 is givenjn Patwardhan et al., Oncogene. 6:917 (1991).
  • Mammalian EGR is a transcription factor that stimulates the activity of a number of mammalian genes and inhibits other genes.
  • the mammalian EGRs contain three zinc fingers of the Cy ⁇ Fu ⁇ class that binds to the GCE site in 5* enhancer region, which has the sequence GCGGGGGCG (SEQ ID NO:7), GCGTGGGCG (SEQ ID NO:8) or GCGAGGGCG (SEQ ID NO:9).
  • GCGGGGGCG SEQ ID NO:7
  • GCGTGGGCG SEQ ID NO:8
  • GCGAGGGCG GCGAGGGCG
  • Egr-1 is constitutively expressed in differentiated embryonal carcinoma (EC) cells where evidence supports its role in maintaining the differentiated state. When EC cells are induced to differentiate, the expression of Egr-1 increases markedly.
  • Egr-1 stimulates the activity of the GCE site in several promoters in transient expression assays.
  • Genes whose transcription has been shown to be inhibited by Egr-1 include the PDGF-A gene (as tested in NTH-3T3 cells), adenosine deaminase (ADA) gene 1 (as tested in murine Cl-ID cells), and the midkine (MK) gene (as tested in PI 9 cells).
  • PDGF-A gene as tested in NTH-3T3 cells
  • ADA adenosine deaminase
  • MK midkine
  • Egr-1 can compete with Spl in binding to an overlapping consensus binding motif in the promoter region of murine ADA therefore abolishing the function of Sp 1.
  • Egr- 1 stimulates the activity of the GC-rich DNA-binding element (GCE) while WT1 inhibits. Madden et al., Science 253: 1550 (1991).
  • Minor modifications can be made to EGR while retaining the growth inhibitory activity of the molecule.
  • Minor modifications include simple substitutions, additions or deletions.
  • Simple substitutions include the substitution of an amino acid for another having a side chain extending from the alpha carbon of the same class, i.e., non-polar (hydrophobic), neutral, positively charged or negatively charged. These modifications may be introduced deuoerately through site-directed mutagenesis, or may be accidental, such as through mutation in hosts having DNA encoding these polypeptides. Any such modified protein can be easily tested for activity in the assays described herein.
  • Fragments of EGR that contain the zinc finger domain useful in the present invention also include the zinc finger domain that extends from amino acids 320 to 431 (U.S. Patent No.
  • EGR4 (Patwardham et al., supra): RGGKCSTRC FCPRPHAKAFA CPNESCVRS FARSDEL ⁇ RH LRIHTGHKPF QCRICLR ⁇ FS RSDHLTTHVR THTGEKPFAC DNCGRRFARS DEKKRHSKNH LRQKARAEER (SEQ ID NO: 10).
  • the zinc finger domain of other mammalian EGRs can be determined by inspecting their amino acid sequences and comparing them with the domains just described.
  • Patent No. 5,837,692 to Mercola et al., issued November 11, 1998) has greater inhibitory effect than the zinc finger alone.
  • the polypeptide fragments and the nucleic acid sequences encoding them have the zinc finger domain of a mammalian EGR, but exclude the "repression domain.”
  • the polypeptide fragments and the nucleic acid sequences encoding them consist essentially of the zinc finger domain and the remainder of the carboxy terminal end of the molecule.
  • the methods of the present invention use a nucleic acid encoding a mammalian EGR, a nucleic acid sequence encoding a fragment of a mammalian EGR comprising the zinc finger domain or a nucleic acid sequence that hybridizes to any of the foregoing nucleic acid sequence under standard hybridization conditions and that encodes a polypeptide having the activity of increasing the expression of at least one anti-metastatic factor in a tumor cell or interfering with the metastasis of a tumor cell.
  • nucleic acid molecules can be present in a vector, such as a viral vector or a liposome, or be present as naked DNA, such as linear DNA or plasmid DNA or other delivery vehicle know in the art.
  • the nucleic acid encoding a mammalian EGR or a portion thereof can be linked to sequences that can directed homologous recombination into the genome of a host cell at desired locations, such as to be regulated by endogenous expression control sequences, such as promoters, using methods known in the art (WO 94/24301 to Smith et al., published October 27, 1994). Such nucleic acids can also be randomly integrated or targeted into the genome of a host cell (see, WO 98/13353 to Whitney, published April 2, 1998, and Skarnes et al., Genes and Development 6:903-918 (1992)). Alternatively, these nucleic acid molecules can be operatively linked to expression control sequences that are operable in a target cell. According to one embodiment of the present invention, the nucleic acid sequence encodes essentially the zinc finger domain of a mammalian EGR.
  • nucleic acid molecule "encodes" a polypeptide if transcription of the nucleic acid molecule and translation of the mRNA produce the polypeptide.
  • nucleic acid molecules of the present invention include those whose nucleotide sequence encodes a polypeptide directly, such as cDNA, or whose nucleotide sequence includes introns that are removed upon transcription into mRNA, such as genomic DNA. It also includes nucleic acid molecules having sequences which are degenerate versions of any of the aforementioned nucleotide sequences.
  • standard hybridization conditions refers to salt and temperature conditions substantially equivalent to 5 X SSC and 65° C for both hybridizations and washes.
  • the activity of any such DNA sequence can be tested by the assays described in the Examples.
  • the nucleic acid molecules of the present invention can be produced by organic synthesis on a commercial nucleic acid synthesizer or through PCR of a nucleic acid encoding a polypeptide useful in the present invention.
  • Nucleic acid sequences encoding mammalian EGR can be identified by probing cDNA libraries under standard hybridization conditions with probes derived from mouse Egr-1, human EGR-1, human EGR2, humanEGR3 or human EGR4 and by analyzing cDNA expression libraries with antibodies against a mammalian EGR.
  • EGR from these mammals can be isolated and partially sequenced, and the sequence can be used to make sets of degenerate nucleic acid probes for probing gene libraries. Other methods for identifying and isolating genes are also known.
  • the expression vectors of this invention have an expression control sequence operatively linked to a nucleic acid molecule of the present invention.
  • An expression control sequence is operatively linked to a nucleic acid molecule when it directs the transcription and translation of that molecule in an appropriate host cell. This includes provision of appropriate start and stop codons.
  • expression of EGR is constitutive.
  • Expression vectors in which the expression control sequence comprises a RSV or CMV promoter will express EGR constitutively. Both promoters are commonly used in the art for the expression or recombinant nucleic acid molecules (Gorman et al., Molec. and Cell Biol., 2: 1044 (1982) and Boshant et al., Cell 41:421 (1985)).
  • Vectors useful in this invention include those capable of transferring genes into mammalian cells. These include viral vectors such as, but not limited to, retroviruses, adenoviruses and adeno-associated viruses, plasmid vectors, cosmids, liposomes and other delivery vehicles as they are known in the art.
  • viral vectors such as, but not limited to, retroviruses, adenoviruses and adeno-associated viruses, plasmid vectors, cosmids, liposomes and other delivery vehicles as they are known in the art.
  • Retroviral vectors are one example of a vector useful for increasing the expression of at least one anti-metastatic factor in a tumor cell or interfering with the metastasis of a tumor cell by gene therapy.
  • a retroviral packaging cell line such as PA317 (ATCC NO: CRL 9078) is used to create infective amphotropic retroviral vectors
  • the retroviral plamid pLNCX can contain the expression control sequence operatively linked to the nucleic acid sequence to be expressed.
  • That plasmid contains a Maloney murine leukemia virus LTR promoter/enhancer (L); neomycin resistance gene encoding neomycin phosphotransferase (N); a human cytomegalic virus LTR/enhancer (C) and the coding gene to be expressed (X).
  • L Maloney murine leukemia virus LTR promoter/enhancer
  • N neomycin phosphotransferase
  • C human cytomegalic virus LTR/enhancer
  • X coding gene to be expressed
  • Retroviruses such as pLHCC (pLHCX in which X is the C domain of Egr-1) or pLHCCD (pLHCX in which X is the CD domain of Egr-1) can be used directly to treat tumors.
  • tumors can be treated with irradiated packaging cells that express these retroviruses (see U.S. Patent No. 5,837,692 to Mercola et al., issued November 17, 1998).
  • Vectors can be administered to a tumor cell in culture by simply adding the vector to the culture or by other means such as ballistics, electroporation or lipofection methods known in the art.
  • Vectors can be administered to a tumor cell in a whole organism by a variety of methods, such as systemic administration, direct injection into a tumor or injection into a locus ⁇ djacent to the tumor, or electroporation directly into a tumor or adjacent thereto, or ballistic delivery to the tumor or adjacent thereto.
  • Vectors can be administered to whole organisms that have naturally occurring tumors or tumors that arise from tumor cells injected or otherwise transplanted into the organism using known methods (see, U.S. Patent No. 5,837,692 to Mercola et al., issued November 17, 1998).
  • the route of administration, dose and regiment of administration for such vectors can be determined as set forth herein for other pharmaceutical compositions.
  • the methods of the present invention operate to increase the expression of at least one anti-metastatic factor in a tumor cell, including but not limited to plasminogen activator inhibitor- l , transforming growth factor beta-1 , and fibronectin.
  • Increased expression means that the expression of mRNA, protein, or modified protein (such as, for example, by glycosylation) is above the natural or normal expression.
  • the increased expression of such anti-metastatic factors can be determined using the methods set forth in the Examples.
  • the methods of the present invention can interfere with the metastasis of a tumor cell, which can be measured using the methods set forth in the Examples.
  • the methods of the present invention also operate to inhibit the growth of a tumor cell.
  • To inhibit the growth of a tumor cell means to decrease the rate at which such a tumor cells replicates, or to decrease the number of living tumor cells in a sample.
  • a sample can De at least one cell in culture or within an animal, such as a mammal, including test animals, such as mice, or humans.
  • the inhibition of such growth as compared to appropriate control conditions and cells can be measured using methods known in the art.
  • the growth and replication of cells can be monitored by microscopic examination, instrumentation counting or viability staining using suitable stains or dyes.
  • the inhibition of such growth can be measured by the reduced increase in tumor mass or volume, or reduced tumor mass or volume.
  • Such rumor mass or volume can be measured by established methods, such as visual examination, excising and weighing or measuring the volume of the tumor, or by imaging methods, such as x-rays or MRI instrumentation.
  • Tumor cells that are administered a vector of the present invention preferably do.not express a detectable level of EGR.
  • the tumor cells express between about 0 and about 10% of the level of EGR normally expressed by these cells, and more preferably between about 2% and about 8%.
  • the present invention also includes a method for identifying compounds or compositions that increase or decrease the expression of an EGR, increase or decrease the expression of at least one anti-metastatic factors in a cell, or increase or decrease cell attachment to substrate.
  • this method includes providing a cell that expresses or does not express EGR or at least one anti-metastatic factors.
  • Such cells can be in culture or in an organism, such as a mouse or other test animal, as a result natural generation of such cells, induced generation of such cells, transgenic manipulation of the organism or by injecting such cells into the organism Using methods known in the art.
  • the cell can either express EGR or at least one anti-metasratic factors endogenously, or express EGR or at least one anti-metastatic factor in response to at least one transfected nucleic acid molecule of the present invention that encode an EGR or a portion thereof.
  • the cells or organism are contacted with a test compound.
  • the resulting increase or decrease of expression of EGR or increased or decreased expression of at least one anti- metastatic factors in the cell is then measured using appropriate methods, such as those described in the Examples, such as detecting EGR mRNA in the cell or EGR in the cell using established methods, such as PCR or immunological methods.
  • Reporter genes can also be used to monitor the expression of a desired gene (see, Whitney et al.,WO 98/13353 to Whitney et al., . published April 2, 1998; WO 94/24301 to Smith et al., published October 3, 1994; and Skarne ⁇ et.al., Genes and Development, 6:903-918 (1992).
  • the increase or decrease of at least one anti-metastatic factor can also be measured using such methods.
  • the attachment of such cells to substrates, such as untreated plastics, or surfaces treated with plasminogen activator inhibitor- 1 or fibronectin can be monitored using the methods of the present invention to evaluate the ability of a test compound to modulate cell metastasis or cell mobility.
  • Test compounds that increase the expression of EGR or at least one anti-metastatic factor are presumptive therapeutic compounds that can treat a tumor or cancer or interfere with the metastasis of a tumor cell.
  • Test compounds that decrease the expression of EGR or at least one anti-metastatic factor are compounds that presumptive therapeutic compounds that can increase ceil mobility.
  • Test compounds that increase cell attachment to a substrate are presumptive therapeutic compounds that can treat a tumor or interfere with metastasis of a tumor.
  • Test compounds that decrease cell attachment to a substrate are presumptive therapeutic compounds that increase cell mobility.
  • test compound The structure of a test compound can be determined or confirmed by methods known in the art, such as mass spectroscopy. For test compounds stored for extended periods of time under a variety of conditions, the structure, activity and potency thereof can be confirmed. Identified test compounds can be evaluated for a particular activity using recognized methods and those disclosed herein. For example, if an identified test compound is found to have anticancer cell activity in vitro, then the bioactive compound or bioactivity would have presumptive pharmacological properties as a chemotherapeutic to treat cancer. Such nexuses are known in the art for several disease states, and more are expected to be discovered over time. Based on such nexuses, appropriate confirmatory in vitro and in vivo models of pharmacological activity, and toxicology, and be selected and performed. The methods described herein can also be used to assess pharmacological selectivity and specificity, and toxicity.
  • test compounds can be evaluated for toxicological effects using known methods (see, Lu, Basic Toxicology, Fundamentals, Target Organs, and Risk Assessment, Hemisphere Publishing Corp., Washington (1985); U.S. Patent Nos; 5,196,313 to Culbreth (issued March 23, 1993) and 5,567,952 to Benet (issued October 22, 1996)).
  • toxicology of a test compound can be established by determining in vitro toxicity toward a cell line, such as a mammalian, for example human, cell line.
  • Test compounds can be treated with, for example, tissue extracts, such as preparations of liver, such as microsomal preparations, to determine increased or decreased toxicological properties of the test compound after being metabolized by a whole organism.
  • the results of these types of studies are predictive of toxicological properties of chemical s in animals, such as mammals, including humans.
  • the toxicological properties of a test compound in an animal model such as mice, rats, rabbits, dogs or monkeys, can be determined using established methods (see, Lu, supra (1985); and Creasey, Drug Disposition in Humans, The Basis of Clinical Pharmacology, Oxford University Press, Oxford (1979)).
  • an animal model such as mice, rats, rabbits, dogs or monkeys
  • the skilled artisan would not be burdened to determine appropriate doses, LD 50 values, routes di administration and regimes that would be appropriate to determine the toxicological properties of the test compound.
  • test compound can be established using several art recognized methods, such as in vitro methods, animal models or human clinical trials (see, Creasey, supra (1979)). Recognized in vitro models exist for several diseases or conditions. For example, the ability of a test compound to extend the life-span of HJN-infected cells in vitro is recognized as an acceptable model to identify chemicals expected to be efficacious to treat HIN infection or AIDS (see, Daluge et al., Antimicro. Agents Chemother. 41 : 1082-1093 (1995)).
  • CsA cyclosporin A
  • acceptable animal models can be used to establish efficacy of test compounds to treat various diseases or conditions.
  • the rabbit knee is an accepted model for testing agents for efficacy in treating arthritis (see, Shaw and Lacy, J. Bone Joint Surg. (Br.) 55: 197-205 (1973)).
  • Hydrocortisone which is approved for use in humans to treat arthritis, is efficacious in this model which confirms the validity of this model (see, McDonough, Phys. Ther. 62:835-839 (1982)).
  • the skilled artisan can be guided by the state of the art to choose an appropriate model, doses and route of administration, regime and endpoint and as such would not be unduly burdened.
  • Selectivity is evident, for example, in the field of chemotherapy, where the selectivity of a chemical to be toxic towards cancerous cells, but not toward non-cancerous cells, is obviously desirable.
  • Selective modulators are preferable because they have fewer side effects in the clinical setting.
  • the selectivity of a test compound can be established in vitro by testing the toxicity and effect of a test compound can be estabhshed in vitro by testing the toxicity and effect of a bioactive compound or bioactivity on a plurality of cell lines that exhibit a variety of cellular pathways and sensitivities.
  • the data obtained form these in vitro toxicity studies can be extended to animal model studies, including human clinical trials, to determine toxicity, efficacy and selectivity of a test compound bioactive compound or bioactivity.
  • test compounds can be often improved by generating additional test chemicals based on the structure/property relationship of a test compound originally identified as having activity.
  • Test compounds can be modified to improve various properties, such as affinity, life-time in blood, toxicology, specificity and membrane permeability.
  • Such refined test compounds can be subjected to additional assays as they are known in the art or described herein. Methods for generating and analyzing such compounds or compositions are known in the art, such as U.S. Patent No. 5,574,656 to Agrafiotis et al.
  • Pharmaceutical compositions are known in the art, such as U.S. Patent No. 5,574,656 to Agrafiotis et al.
  • the present invention also encompasses a test compound in a pharmaceutical composition
  • a pharmaceutically acceptable carrier prepared for storage and preferably subsequent administration, which have a pharmaceutically effective amount of the test compound in a pharmaceutically acceptable carrier or diluent.
  • Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co., (A.R. Gennaro edit. (1985)).
  • Preservatives, stabilizers, dyes and even flavoring agents can be provided in the pharmaceutical composition.
  • sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid can be added as preservatives.
  • antioxidants and suspending agents can be used.
  • test compounds of the present invention can be formulated and used as tablets, capsules or elixirs for oral administration; suppositories for rectal administration; sterile solutions, suspensions or injectable administration; and the like.
  • injectables can be prepared in conventional forms either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Suitable excipients are, for example, water, saline, dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate, cysteine hydrochloride and the like.
  • the injectable pharmaceutical compositions can contain minor amounts of nontoxic auxiliary substances, such as wetting agents, pH buffering agents and the like.
  • absorption enhancing preparation such as liposomes
  • the pharmaceutically effective amount of a test compound required as a dose will depend on the route of administration, the type of animal or patient being treated, and the physical characteristics of the specific animal under consideration. The dose can be tailored to achieve a desired effect, but will depend on such factors as weight, diet, concurrent medication and other factors which those skilled in the medical arts will recognize.
  • the pharmaceutical compositions can be used alone or in combination with one another, or in combination with other therapeutic or diagnostic agents. These products can be utilized in vivo, preferably in a mammalian patient, preferably in a human, or in vitro.
  • the pharmaceutical compositions can be administered to the patient in a variety of ways, including parenterally, intravenously, subcutaneously, intramuscularly, colonically, rectally, nasally or intraperiotoneally, employing a variety of dosage forms. Such methods can also be used in testing the activity of test compounds in vivo.
  • the useful in vivo dosage to be administered and the particular mode of administration will vary depending upon the age, weight and type of patient being treated, the particular pharmaceutical composition employed, and the specific use for which the pharmaceutical composition is employed.
  • the determination of effective dosage levels can be accomplished by one skilled in the art using routine methods as discussed above.
  • human clinical applications of products are commenced at lower dosage levels, with dosage level being increased until the desired effect is achieved.
  • acceptable in vitro studies can be used to establish useful doses and routes of administration of the test compound.
  • the dosage for the test compound of the present invention can range broadly depending upon the desired affects, the therapeutic indication, route of administration and purity and activity of the test compound.
  • dosages can be between about 1 ng/kg and about 10 ng/kg, preferably between about 10 ng/kg and about 1 mg/kg, more preferably between about 100 ng/kg and about 100 micrograms/kg, and most preferably between about 1 microgram/kg and about 10 micrograms/kg.
  • the exact formulation, route of administration and dosage can be chosen bv the individual physician in view of the patient's condition (see, Fingle et al., in The Pharmacological Basis of Therapeutics (1975)). It should be noted that the attending physician would know how to and when to terminate, interrupt or adjust administration due to toxicity, organ dysfunction or other adverse effects. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate.
  • the magnitude of an administrated does in the management of the disorder of interest will vary with the severity of the condition to be treated and to the route of administration.
  • the severity of the condition may, for example, be evaluated, in part, by standard prognostic evaluation methods.
  • the dose ana perhaps dose frequency will also vary according to the age, body weight and response of the individual patient, including those for veterinary applications.
  • compositions can be formulated and administered systemically or locally.
  • Techniques for formation and administration can be found in Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, PA (1990), Suitable routes of administration can include oral, rectal, transdermal, otic, ocular, vaginal, transmucosal or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.
  • the pharmaceutical compositions of the present invention can bejformulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution or physiological saline buffer.
  • physiologically compatible buffers such as Hanks' solution, Ringer's solution or physiological saline buffer.
  • penetrans appropriate to the barrier to be permeated are used in the formulation.
  • Such penetrans are generally known in the art.
  • Use of pharmaceutically acceptable carriers to formulate the pharmaceutical compositions herein disclosed for the practice of the invention into dosages suitable for systemic administration is within the scope of the invention.
  • the compositions of the present invention in , particular, those formulation as solutions, can be administered parenterally, such as by intravenous injection.
  • compositions can be formulated readily using pharmaceutically acceptable carriers well known in the art into dosages suitable for oral administrations.
  • Such carriers enable the bioactive compounds and bioactivities of the invention to be formulated as tables, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
  • Agents intended to be administered intracellularly may be administered using techniques well known to those of ordinary skill in the art. For example, such agents may be encapsulated into liposomes, then administered as described above. Substantially all molecules present in an aqueous solution at the time of liposome formation are incorporated into or within the liposomes thus formed. The liposomal contents are both protected from the external micro-environment and, because liposomes fuse will cell membranes, are efficiently delivered into the cell cytoplasm. Additionally, due to their hydrophobicity, small organic molecules can be directly administered intracellularly.
  • compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose. Determination of the effective amount of a pharmaceutical composition is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
  • these pharmaceutical compositions can contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active chemicals into preparations which can be used pharmaceutically.
  • suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active chemicals into preparations which can be used pharmaceutically.
  • the preparations formulated for oral administration may be in the form of tables, dragees, capsules or solutions.
  • compositions of the present invention can be manufactured in a manner that is itself known, for example by means of conventional mixing, dissolving, granulating, dragee-making, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • Pharmaceutical formulations for parenteral administration include aqueous solutions of active chemicals in water- soluble form. Additionally, suspensions of the active chemicals may be prepared as appropriate oily injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides or liposomes.
  • Aqueous injection suspensions may contain substances what increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran.
  • the suspension can also contain suitable stabilizers or agents that increase the solubility of the chemicals to allow for the preparation of highly concentrated solutions.
  • compositions for oral use can be obtained by combining the active chemicals with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tables or dragee cores.
  • suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannit ⁇ or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone.
  • disintegrating agents can be added, such as the cross-linked polyvinyl pyrolidone, agar, alginic acid or a salt thereof such as sodium alginate.
  • Dragee cores can be provided with suitable coatings. Dyes or pigments can be added to the tablets or dragee coatings for identification or to characterize different combinations of active doses.
  • test compounds of the present invention and pharmaceutical compositions that include such test compounds are useful for treating a variety of ailments in a patient, including a human.
  • the test compounds of the present invention have anti-metasiatic effects, anti-tumor effects and anti-cancer effects.
  • a patient in need of such treatment can be provided a test compound of the present invention, preferably in a pharmacological composition in an effective amount to reduce metastasis, reduce the growth rate of tumors, reduce to tumor mass of tumors or treat cancer or carcinomas.
  • the amount, dosage, route of administration, regime and endpoint can all be determined using the procedures described herein or by appropriate government agencies, such as the United Stated Food and Drug Administration. Examples
  • Fibrosarcoma HT1080 subione H4 cells ATCC NO: ), ERG- 1 -expressing transfectants (H4E2 (ATCC NO: ), H4E3 (ATCC NO: ) and H4E9
  • H4E4 H4E6 (ATCC NO: )
  • H4E4 mouse wild-type erg-1
  • H4E6 H4E6
  • Cells were initially cultured at a density of about 4 x 10 4 cells/cm 2 , incubated overnight, washed twice with ice-cold phosphate-buffered saline (PBS), and lysed by scraping using RIPA buffer (1% ⁇ onidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 100 ⁇ g/ml phenylmethylsulfonyl floride, 1 mM aprotinin and 1 M sodium orthovanadate). The lysates were passed though a 21 -gauge needle to shear D ⁇ A in the lysates, incubated for one Hour on ice and centrifuged at 12,000 x g for twenty minutes and the supematants collected.
  • PBS ice-cold phosphate-buffered saline
  • RIPA buffer 1% ⁇ onidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 100 ⁇ g/ml phenylmethylsulf
  • Protein concentration in the supematants were determined using Bio-Rad protein assay reagent. Samples of the supematants containing 100 ⁇ g of protein were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using a 7% gel. Protein in the gel was electrophoretically transferred onto polyvinylidene difluoride membrane (Millipore Corporation, Bedford, Mass) and incubated with polyclonal rabbit anti-Egr-1 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA). A secondary antibody, anti-rabbit IgG antibody with an appropriate label. Immunpreactive bands on the membrane were visualized by enhanced chemiluminescence (ECL, Amersham
  • plasminogen activator inhibitor PAI-1
  • 2 x 10 5 cells were initially placed in standard 6-well tissue culture plates in DMEM supplemented with 5% fetal bovine serum and incubated overnight. These cells were exposed to cysteine/methionine-free DMEM in the presence or absence of various concentrations (between about 0.001 ng/ml and about 100 ng/ml) of recombinant human TGF- ⁇ l (rhTGF- ⁇ 1) (R&D Systems Inc., Minneapolis, MN) for about 30 ⁇ g/ml of mouse monoclonal anti-TGF- ⁇ 1A3 (Genzyme Corp., Cambridge, MA) for two hours. The cells were then exposed to 35 S-cysteine/methionine at 50 ⁇ Ci/ml of (1180 Ci mmol; Trans-
  • fibronectin (FN) assay 2 x 10 s cells were initially cultured in standard 6-well tissue culture plates and treated overnight with or without 10 ng/ml of TGF- ⁇ l or 30 ⁇ g/mLof mouse monoclonal anti-TGF- ⁇ , w cysteine/methionine-free media. The media was collected and subjected to absorption of fibronectin using gelatin-Sepharose beads (Amersham Pharmacia Biotech) in the presence of 0.5% Triton X- 100 as previously described (Carcamo et al, Mol. Cell. Biol. 14:3810-3821 (1994)).
  • hTGF- ⁇ l antisense oligonucleotide corresponding to the human TGF- ⁇ l mRNA (hTGF- ⁇ l antisense oligonucleotide) and a scrambled sequence control sequence (5'-GTCCCTATACGAAC-3' (SEQ ID NO:2)) were synthesized by established methods.
  • the hTGF- ⁇ l antisense oligonucleotide has been shown to specifically reduce TGF- ⁇ l expression (Paulus et al., J. Neuropathol. Exp. Neurol. 54:236-244 (1995)).
  • Cationic liposome-mediated transfection was used to introduce oligonucleotides into fibrosarcoma HT1080 subclone H4 cells, or the erg-J transfected clones using established methods (Suzuki et al., Mol. Cell. Bio. 18:3010-3020 (1988)). Briefly, oligonuleotides were dissolved in one volume of antibiotic-free medium and mixed with LipofectinTM reagent (Life Technologies, Inc.) dissolved in the same volume of antioiotic-free medium and incubated for fifteen minutes at room temperature.
  • oligonucleotide-iiposome complexes thus formed were diluted with four volumes of antibiotic-free medium and added to cells that had be cultured to about 60% confluence and previously washed twice with antibiotic- free medium.
  • concentration of oligonuleotides and LipofectinTM in the transfection was 1 mM and 1%, respectively.
  • fresh normal growth medium containing 5% fetal bovine serum as added to the cells.
  • Forty-eight hours later the cells were analyzed for expression of plasminogen activator inhibitor- 1 (PAI-1) or fibronectin (FN).
  • PAI-1 plasminogen activator inhibitor- 1
  • FN fibronectin
  • Nuclear extracts of clone H4H9 (ERG-1 expressing), clone H4 (non-ERG-1 expressing) and clone H4N (non-ERG-1 expressing) were prepared using established methods (Brown et al., Eur. J. Immunol. 22:2419-2420 (1992)). Protein concentrations in these nuclear extracts were determined using protein assay reagents (BioRad). Synthetic double-stranded oligonuleotides bearing sequences corresponding to either -75 to -52 base pairs of human FN promoter (Site A) or -4 to +18 base pairs of human FN promoter (Site B) (Brown et al., Eur. J. Immunol.
  • the DNA sequence for Site A is 5'- GATCTCTCTCCTCCCCCGCGCCCCGGGG-3' (SEQ ID NO:3) with a prototypical EGR-1 binding site of 5'-CTCCCCCGC-3' (SEQ ID NO:4).
  • the DNA sequence for Site B is 5'- GATCTCCGACGCCCGCGCCGGCTGTG-3 • (SEQ JD NO: 5) with a prototypical ERG-1 binding site of 5'-CGCCCGCGC-3' (SEQ ID NO:6).
  • the full-length oligonucleotides for Site A and Site B were end-labeled with [ ⁇ - 32 P]ATP using T4 polynucleotide kinase using established methods. These labeled oligonuleotides were uses as "Probe A” and "Probe B.”
  • Gel shift assays were performed by incubating 20 ⁇ g of nuclear extract with Probe A or Probe B (1 x 10 5 cpm) for twenty minutes at 4°C in a 20 ⁇ L reaction volume containing 25 mM HEPES buffer, pH 7.9, 60 mM KC1, 2 mM MgCl 2 , 0.1 mM EDTA, 0.5 mM dithiothreitol, 100 ⁇ g/ml spermidine, 10% glycerol and 100 ⁇ g/ml bovine serum albumin. Protein-Probe complexes were separated from free Probe by electrophoresis through a 6% nondenaturing acrylamide gel in 0.5X Tris borate/EDTA buffer.
  • Antibody supershift experiments were performed adding antibody specific for Spl (Santa Cruz Biotechnology, Santa Cruz, CA) or rabbit polyclonal Egr-1 antiserum (Huang et al., Oncogene 9 1367-1377 (1994)) to the reaction mixture before the appropriate radiolabeled Probe A, Probe B, or both were added.
  • recombinant GST-ERG- 1 fusion protein or wild-type FLAG-tagged-S348A/S350A-EGR-l and FLAG-tagged-ERG-l ⁇ S348A/S350A mutant fusion proteins were used as controls.
  • the FLAG-tagged proteins are fusion proteins produced analogues to GST-EGR proteins. Mutants were introduced into the DNA binding sites of Erg- 1 using the Quick Change method using polymerase chain reaction primers comprising the
  • Cell adhesion assays were performed using ELISA plates (Sarstedt Inc., Newton, NC) or 35mm polystyrene Petri dishes (Falcon, Becton Dickinson, Bedford, MA). Cell adhesion assays performed in Petri dishes were performed by adding about 3 x 10 4 cells/cm 2 of surface area. After incubation for between about four and about 7 hours, cells that were not attached to the surface were washed away using PBS at 37°C. Adherent cells were harvested from the surface using trypsin and counted using a Coulter counter.
  • ELISA plates were pretreated by coated with 5 ⁇ g/ml or 0.25 ug/ml of human plasma fibronectin (Boehringer Mannheim) or 5 ⁇ g/ml recombinant active human PAI-1 (American Diagnostica, Inc. Greenwich, CT) for one hour at 37°C. Non-specific sites on the pretreated plates were blocked using 0.1% bovine serum albumin in PBS. Cells were dispensed into the wells at a density of about 4 x 10 4 cells/well. After incubation of a variety of times, any non-adherent cells were washed away with gentle washing with warm PBS.
  • Adherent cells were stained with 1 % crystal violet in 20% methanol for fifteen minutes, washed with distilled water, and solubilized with 2% SDS. The absorbance of the solution in the wells at 590 nm was determined using a microtiter plate reader.
  • PAI-1 neutralization 10 ⁇ g/ml of monoclonal antibody anti-human PAI-1 (American Diagnostics) were added to the wells at with cells and the effect on cell adhesion was observed.
  • GRGDSP and GRGESP peptides were added to the wells at about 10 ⁇ g/ml and about 50 ⁇ g/ml with the cells and the effect on cell adhesion was observed.
  • Example 2 Effect of EGR-1 on the Expression of PAI-1 in Fibrosarcoma Cells
  • fibrosarcoma HT 1080 subclone H4 cells stably transfected with an expression vector for wild-type EGR-1 were used (Huang et al., Cancer Res. 55:5054-5062 (1995)), A series of transfectants of HT 1080 subclone H4 that express varying amounts of EGR- 1 were used to determine whether PAI-1 or FN was synthesized and secreted in proportion to the amount of EGR-1 expressed by these cells.
  • the relative expression of EGR-1 by these cells as confirmed by Western blot analysis, as is shown in FIG. 1 A.
  • H4E4, H4E6, parental H4 and empty vector control cells H4N did not express detectable levels of EGR-1
  • the clones were labeled with [ 35 S]cysteine/methionine in the presence or absence of 10 ng/ml of recombinant human TGF- ⁇ l (rhTGF- ⁇ 1) for four hours.
  • the resultant expression of PAI-1, if any, was monitored by electrophoresis and autoradiography (see, FIG. IB).
  • All of the clones tested exhibited a significant (p ⁇ 0.05) increase in the secretion of PAI-1 following treatment with rhTGF- ⁇ 1 (see, FIG. IC).
  • the total PAI-1 measured for each clone correlated with basal PAI-1 expression in these cells observed in the absence of stimulation with rhTGF- ⁇ 1 (see, FIG. IC).
  • Example 3 Induction of Fibronectin Expression in Fibrosarcoma cells by EGR-1.
  • the levels of FN secreted by the EGR-1 expressing and EGR-1 non-expressing cells were determined by metabolically labeling the cells for two hours.
  • the labeled FN produced by the cells and presented in the culture medium was absorbed from the culture medium with gelatin- Sepharose beads known to bind FN (Scott et al., J. Immunol. Methods 43:29-33 (19si)).
  • the absorbed FN was recovered and analyzed by SDS-PAGE.
  • the characteristic band of FN at 220 KDa was observed at maximum intensity in the medium for H4E9 cells and band was noted in samples from H4E2 cells and H4E3 cells, with a band intensity between H4E9 cells, parental cells, and cells that do not express EGR-1 (see, FIG.
  • FN can be stimulated by TGF- ⁇ l in prostatic carcinoma cells (Frazen et al., Exp. Cell Res. 207: 1-7 (1993)), colon cancer cells (Huang et al., Int. J. Cancer 261 :4337- 4345 (1986)) and lung mink MvlLu cells (Laiho et al. Mol. Cell. Bio. 11:972-978 (1991)).
  • prostatic carcinoma cells Frazen et al., Exp. Cell Res. 207: 1-7 (1993)
  • colon cancer cells Huang et al., Int. J. Cancer 261 :4337- 4345 (1986)
  • lung mink MvlLu cells Laiho et al. Mol. Cell. Bio. 11:972-978 (1991)
  • EGR-1 -induced TGF- ⁇ l regulates the expression of FN in , fibrosarchoma cells
  • the H4 clones of the present application were contacted with rhTGF-
  • FN is preferentially regulated by TGF- ⁇ l .
  • the high correlation between EGR-1 expression and FN expression without the role of TGF- ⁇ l suggests, as one possible mechanism that the inventors expressly do not wish to be bound to, that EGR-1 directly regulates the expression of the FN gene.
  • Example 5 The Requirement of TGF- ⁇ l for the Secretion of PAI-1 and FN in egr-1 Regulated H4 Cells - Immunological Methods.
  • Example 6 The Requirement of TGF- ⁇ l for the Secretion of PAI-1 and FN in egr-1 Regulated H4 Cells - Antisense Methods.
  • Example 5 The results obtained in Example 5 were confirmed using antisense technologies.
  • antisense TGF- ⁇ 1 molecules comprising SEQ ID NO: 1 , or comprising tne control scrambled SEQ ID NO:2, were used as set forth in Example 1.
  • Nucleic acid molecules comprising SEQ ED NO: 1 have been used to reduce the transcription levels and production of TGF- ⁇ l (LeRoy et al., J. Bio. Chem. 271: 11027-11033 (1996) and Paulus et al., J. Neuropathol. Exp. Neurol. 54:236-244 (1995)).
  • SEQ ID NO: 1 or SEQ ID NO:2 see, FIG. 5).
  • the level of PAI-1 in EGR-1 expressing H4E9 cells transfected with SEQ ID NO: 1 was reduced by over 75% in EGR-1 producing cell lines to levels of PAI-1 in control cells H4 and H4N in H4E9.
  • the level of PAI-1 in EGR-1 expressing cells was not influenced by transfection with the scrambled SEQ ID NO:2 or by lipofection reagents alone (see, FIG.5).
  • the expression of FN was riot inhibited in cells expressing EGR-1 by transfection of either SEQ ID NO: 1 or SEQ ID NO.2 (see, FIG. 5).
  • Example 7 Binding of Nuclear and Recombinant EGR-1 to the Proximal Region of the
  • Electrophoretic mobility assays were used to determine if EGR-1 binds to either Probe A or Probe B.
  • a recombinant wild-type FLAG-tagged EGR-1 fusion protein and control mutant FLAG-tagged-EGR- 1 ⁇ S348A/A350A fusion protein were sued for these experiments.
  • the FLAG-tagged-EGR- 1 ⁇ S348A/A350A fusion protein is a serine-alanine mutant at position 348 and 350 in the EGR-1 zinc finger domain, thereby reducing DNA binding activity (Wilson et al., J. Biol. Chem. 267:3718-3724 (1992) and Pavletich et al., Science 10:809-817 ( 1991 )) .
  • Example 8 Enhanced Expression of Fibronectin and PAI-1 in EGR-1-Expressing Cells Increases Adherence to a Substratum.
  • H4E9 cells had attached to polystyrene Petri dishes after four hours compared with about 8% for the control cell lines (9% of H4 cells and 7% of H4N cells, p ⁇ 0.0006). After seven hours, 88% of H4E9 cells attached to the polystyrene Petri dishes, whereas only 58% of the control cells H4 or H4N attached to Petri dishes (p ⁇ 0.0001) (see, FIG. 7).
  • adhesion assays in the presence and absence of rhPAI-1 and human plasma FN (see, FIG. 7B).
  • UE-1 cells are transfected clones of U251, which stably expressed pCMV, an empty vector or pCMV/ Egr-1, which carries a full-length mouse Egr-1 cDNA, and are maintained in the presence of 400 ⁇ g/ml G418 (Huang et al,
  • a retroviral vector, pLHCX (Potapova, et al, Cancer Res. 56, 280-286 (1996)). containing the full-length mouse Egr-1 cDNA was transfected into an amphotropic retrovirus producer cell line, PA317. High titer retrovirus producer clones were obtained by growing in the presence of 100 ⁇ g/ml hygromycin B. The medium from the cultured clones containing the retrovirus was harvested from the confluent producer clones, filtered through a 0.45- ⁇ m pore size filter, then incubated with U251 cells for 2 hr in the presence of Polybrene (8 ⁇ g/ml).
  • the membranes were stripped and reprobed with anti- ⁇ -actin (Sigma Chemical Co., St. Louis, MO).
  • anti- ⁇ -actin Sigma Chemical Co., St. Louis, MO.
  • the intensity of the bands was determined by image analysis using a Kodak Digital ScienceTM ID image analysis system (East Kodak Co., Rochester, NY).
  • TGF- ⁇ l cDNA probe was used to measure parental cell U251 and EGR-1- expressing cell clones. Hybridization was performed according to the method of Church et al. (Church and Gilbert Proc. Nail. Acad. Sci. USA 81, 1991-1995, (1984)). To assess lane-loading differences, membranes were stripped and reprobed with an L32-4A cDNA probe, a mouse ribosomal protein. TGF- ⁇ l RNA level was normalized to the respective L32-4A level using densitometry.
  • the number of cells was determined by harvesting and counting (Coulter Counter, Coulter Electronics, FL). In cases of U251 cells stimulating by mitogenic stimuli, the "quiescent" cells were treated with 100 ng/ml PMA, 20% FBS, 40J/m 2 UV for 4 hr, and medium without serum were added and incubated for 24 hr. After 24 hr, medium was collected for TGF- ⁇ l ELISA assay.
  • PAI-1 plasminogen activator inhibitor
  • 2 x 10 5 cells were plated in 6-well tissue culture plates in DMEM supplemented with 5% fetal bovine serum and incubated overnight. Then the medium was removed and the cells were subjected to cysteine-methionine-free DMEM in the absence or presence of 30 ⁇ g/ml monoclonal mouse anti-TGF- ⁇ i >2)3 (Genzyme Corp., Cambridge, MA) for 2 hr at which time [ 35 S]- cysteine-methionine was added to 50 ⁇ Ci/ml (1,180 Ci/mmol; Trans- 35 S-label; ICN Biochemicals Inc., Costa Mesa, CA) for an additional 2 hr.
  • PAI-1 plasminogen activator inhibitor
  • Extracellular matrix was prepared as described (Der, et al. J. Cell Sci. 52, 151-166, (1981)). Briefly, labeled cell monolayers were rinsed with PBS, and the cytosolic and nuclear proteins were extracted by subsequent washes with hypotonic buffer and sodium deoxycholate. The remaining labeled extracellular matrix proteins were recovered by addition of electrophoresis buffer to the washed wells followed by scraping. The samples were subjected to 10% SDS- PAGE, and the gels were treated with Fluoro-HancerTM autoradiography enhancer
  • FN assay 2 x 10 5 cells were plated in 6-well tissue culture plates. The cells were treated overnight without or with 30 ⁇ g/ml monoclonal mouse anti-TGF- ⁇ 1,2,3 in cysteine-methionine-free media. The next day, [ 35 S]-cysteine-methionine was added to 50 ⁇ Ci/ml for 2 hr. The media were collected and subjected to adsorption on gelatin-Sepharose beads (Pharmacia Biotech Inc., Piscataway, NJ) in the presence of 0.5% Triton X-100 as described (Carcamo, et al. Mol. Cell. Biol. 14, 3810-3821, (1994)). The samples were resolved by 7% SDS-PAGE, and the gels were treated with Fluoro-HancerTM for 30 min followed by drying and autoradiography.
  • the fragment containing the FN promoter region between positions -105 and +18 was cleaved from phFN 105CAT (a gift of Dr. K. Oda) with Bglll and Hind ⁇ ll (Suzuki, et al. Mol. Cell. Biol. 18, 3010-3020, (1998)), inserted into the Bgltt-Hindm sites of pGL3- promoter vector (Promega, Madison, WI) thereby replacing the simian virus 40 (SV40) promoter to generate pGLFN1051uc.
  • phFN 105CAT a gift of Dr. K. Oda
  • Bglll and Hind ⁇ ll Suzuki, et al. Mol. Cell. Biol. 18, 3010-3020, (1998)
  • UE- 1 parental cells U251 and the EGR- 1 -expressing cloned cells, UE- 1 , were grown in six- well plates at a density of 2 x 10 5 cells/well were treated with Lipofectin (Gibco BRL-Life Tech. Rockville, MD) in the presence of 2 ⁇ g of reporter construct alone or 2 ⁇ g of reporter construct combined with various amounts of expression plasmids.
  • Cells were maintained in the presence of the Lipofectin complexes for 16 hr, washed with PBS, and then grown in DMEM medium containing 5% FBS. After 32 hr of incubation, the cells were harvested and luciferase activity was measured with the Luciferase Assay Kit of Promega (Madison, WI).
  • Nuclear protein extracts were prepared from the clone of maximum EGR-1 expression (UE-1) and from non-expressing cells (U251, UN) as described (Dean et al., supra). The protein concentrations in the nuclear extracts were determined by protein assay kit (Bio-Rad Lab., Hercules, CA). Synthetic double-stranded oligonucleotides bearing sequences corresponding to either -75 to -52 bp or -4 to +18 bp of the human EN promoter, termed GC ⁇ -"A" and GCE-"B" respectively, were selected based on an analysis of the sequence of the human EN promoter region for the presence of GC ⁇ s (Transcription Element Search Software).
  • the D ⁇ A sequences for the two oligonucleotides are for GCE-"A", 5'-GATCTCTCTCCTCCCCCGCGCCCCGGGG-3': (SEQ ID NO.: 3) and for GCE-"B", 5'-GATCTCCGACGCCCGCGCCGGCTGTG-3' (SEQ ID NO.: 5).
  • the prototypic EGR-1-binding sites are underlined.
  • oligonucleotides of GCE-"A" and “B” were end-radiolabeled with [ ⁇ - 32 P]-ATP by use of T4 polynucleotide kinase according to the supplier's specification (Pharmacia Biotech Inc., Piscataway, NJ) and used as "probes" A and B.
  • Gel shift assays were performed as follows: nuclear extracts (20 ⁇ g protein) were incubated with radiolabeled DNA probe (1 x 10 5 cpm) for 20 min at 4°C in a 20 ⁇ l reaction containing 25 mM HEPES, pH 7.9, 60 mM KC1, 2 mM MgCl 2 , 0.1 mM EDTA, 0.5 mM dithiothreitol, 100 ⁇ g/ml spermidine, 10% glycerol, and 100 ⁇ g/ml bovine serum albumin. Protein-DNA complexes were separated from free DNA probe by electrophoresis through 6% nondenaturing acrylamide gels in 0.5 x Tris Borate-EDTA.
  • the gels were dried and exposed to X-ray film (Kodak X-OMAT, Kodak Company, Rochester, NY) for autoradiography.
  • X-ray film Kodak X-OMAT, Kodak Company, Rochester, NY
  • excess unlabeled oligonucleotides for probes A and B or oligonucleotides containing two consensus EGR-1 -binding sequences (GCE) and mutated EGR-1 -binding sequences (mGCE) were incubated with the reaction mixture for 15 min at 4°C before the addition of the radiolabeled probes A and B.
  • GCE consensus EGR-1 -binding sequences
  • mGCE mutated EGR-1 -binding sequences
  • the specific antibodies as rabbit polyclonal EGR-1 antiserum Huang, et al. Oncogene 9, 1367-1377, (1994) were added to the binding reactions and incubated for 15 min before the appropriate radiolabeled probe was added.
  • the cells were allowed to attach for 3 hr at 37 °C in a 10% CO 2 incubator. The wells were then washed gently with warm PBS to remove unattached cells. The number of cells attached to the wells was estimated by the tetrazolium-based colorimetric MTS/PMS assay according to the manufacture's instruction (Promega, Madison, WI).
  • EGR-1 protein appears to play important roles in normal brain development and brain injury responses following ischaemia and nerve transection (McCormack, et al. Mol. Brain Res. 12, 215-223, (1992) and Kaplan, et al. Dev. Brain Res. 90, 174-179, (1995)).
  • glioblastoma cell lines U251, T98G, U-373MG, U-87MG. Western blot analysis showed that the basal protein expression of EGR-1 in these cell lines is undetectable whereas basal level expression of EGR-1 in rat primary astrocytes is readily detectable (data not shown). The basis of this effect was explored in detail in these cell lines.
  • EGR-1 expression is nearly undetectable for cells growing in complete medium containing 5%-10% fetal bovine serum or in cells made "quiescent” by culturing in 0.5% serum for 24 hr.
  • Quiescent cells were treated with PMA, 20% fetal bovine serum, or 40 J/m UV for 2 hr, since these stimuli are known to induce both Egr-1 mRNA and EGR-1 protein synthesis (Sokhatme et al, supra and Cao, et al. Mol. Cell. Biol. 10, 1931-1939, (1990)).
  • Egr-1 protein was differentially induced by UV, PMA and serum stimulation.
  • the protein exhibits the characteristic 85 kDa apparent size of full-length normal EGR-1, suggesting that Egr-1 gene transcription and transcription processing remains intact but that steady state level expression is selectively reduced. This led us to investigate the functional effects of re-expression Egr-1 in glioblastoma. Glioblastoma Cell Line U251 Overexpressing EGR-1 Exhibits Elevated
  • nuclear protein extracts from the clones were analyzed by Western blot analysis using antibodies against EGR-1. All three clones (UE-1, UE-21, UE-13) express an 85 KDa protein with broad band distribution characteristic of native EGR-1, which ranges in relative expression levels up to 8.5-fold. Thus, the selected clones exhibit graded levels of EGR-1.
  • EGR-1 inhibits proliferation and transformation of v-sis transformed NIH/3T3 cells (Huang, et al. Oncogene 9, 1367-1377, (1994)), human tumor cell lines such as fibrosarcoma HT1080, osteosarcoma Saos2, glioblastoma U251 and U373, and breast carcinoma ZR75-1 (Huang et al. (1995), supra).
  • EGR-1 directly activates the expression and secretion of active TGF- ⁇ l (Liu, et al. Proc. Natl. Acad. Sci. USA 93, 11831-11836, (1996)).
  • TGF- ⁇ l is an important marker of functional EGR- 1.
  • total RNA was prepared from parental cells U251, empty vector control clones UN, UX-13 and EGR-1 -expressing clones UE-1, UE-21, UE-13.
  • TGF- ⁇ l mRNA A substantial increase of TGF- ⁇ l mRNA was seen in all EGR-1 -expressing cells up to 2.5- fold, as compared to non-EGR-1 -expressing control cells.
  • TGF- ⁇ 1 protein was also elevated in the EGR- 1 -expressing U251 clones, the TGF- ⁇ 1 concentration in conditioned medium from the same cloned lines was measured by the ELISA method.
  • the EGR-1 -expressing clones secreted 2-3 times more active form of TGF- ⁇ l in the medium compared to parental cells (U251) or either of the empty- vector control cell line (UN or UX-13). Similarly, total TGF- ⁇ l (sum of the active form and latent form following acid-activation) was specifically increased.
  • TGF- ⁇ l has been reported to strongly induce the production of extracellular matrix components such as FN and PAI-1 (Ignotz, R. A. and Massague, J. J. Biol. Chem. 261, 4337-45, (1986); Laiho, et al. J. Biol. Chem. 262, 17467-74, (1987) and Massague, J. Annu. Rev. Cell Biol. 6, 597-641, (1990)).
  • FN extracellular matrix components
  • Cell lines were metabolically labeled with [ 35 S]-cysteine/methionine for 2 hr, and the labeled FN was adsorbed from the conditioned medium with gelatin-Sepharose beads that are known to specifically bind FN (Amersham Pharmacia Biotech.) (Liu et al. (1999), supra).
  • Cells, UE-21 and UE-1, that overexpressed EGR-1 exhibited increased FN expression to over 6-fold compared to parental U251 cells and empty vector control cell clones UN, UX-13.
  • UE-13 expressed minimal basal levels of FN protein consistent with the lower expression of EGR-1.
  • the increased expression of FN but not PAI-1 in EGR-1 expressing cells is independent of the enhanced expression of TGF- ⁇ l. Both PAI-1 and FN have been reported to be under regulation of TGF- ⁇ l (Ignotz et al, supra, Laihoetal et al, supra and Massague et al, supra). Thus, the EGR-1 -stimulated increase in TGF- ⁇ l maybe the basis of the elevated expression of FN and/or PAI-1. In order to test whether the increased production of PAI-1 or FN in EGR-1 -expressing cells is due to the elevated secretion amounts of TGF- ⁇ l, we used TGF- ⁇ neutralizing antibodies to specifically block the expression of TGF- ⁇ l.
  • GCEs Greennese-activated FN promoter activity
  • a reporter constructs containing the region spanning positions -105 lo +18 of the human FN promoter, which contains GC-rich sequences This construct was transiently Mercola et al. transfected into the clone with maximum of EGR-1 expression UE-1 and parental cells U251.
  • the EGR-1 -expressing clone UE-1 substantially activated the FN promoter- driven luciferase reporter by 5.5-fold over the same reporter in U251 control cells and 27.5-fold over the reporter control vector pGL, an empty luciferase vector.
  • luciferase activity was not significant different in EGR-1 -expressing cells UE-1 and U251 parental cells. This result confirms that EGR-1 did not augment luciferase activity of the luciferase control vector (pGLvector). In contrast, the luciferase activity was increased about 5-fold in the FN promoter-driven reporter compared to the empty luciferase vector (pGL vector) in the parental cells U251 , suggesting that some additional factor in U251 cells modestly activates the FN promoter.
  • One candidate factor is Sp-1.
  • Sp-1 a transcription factor
  • GC-rich elements Suzuki, et al, supra. If EGR-1 is indeed the major factor responsible for transactivation of the FN gene, it might be expected to exhibit a saturated dose-response for transactivation of the FN luciferase reporter, but not for the empty control vector.
  • These constructs were co- transfected with expression vector for Egr-1 in U251 cells. It was shown that Egr-1 can induce the expression of the FN promoter-driven luciferase reporter.
  • the enhancement of reporter activity is proportional to the quantity of the co-transfected Egr-1 expression vector with a maximum activation of 27-fold. In contrast, there is no systematic activation of the control reporter. Moreover, there is evidence of a saturation effect consistent with a stoichiometric EGR-1 -DNA binding process. Thus, these experiments show that EGR-1 has a potent activating effect on the FN promoter activity that occurs in a dose-dependent and saturable manner. These results also suggest that the regulatory effect of EGR-1 on increased FN secretion is mediated by direct activation of the FN promoter region.
  • EGR-1 binds to EGR-1 -binding sites of human FN promoter region.
  • the luciferase reporter studies showing functional regulation of transcription of FN promoter element by EGR-1 predict that EGR-1 directly and specifically binds to the GC-rich region of the FN promoter.
  • Transcription Element Search Software to screen 742 base pairs of 5 '-flank region (Dean et al, supra) of human FN promoter (exon 1) and identified two potential EGR-1 consensus sites termed GCE-"A" and GCE-"B".
  • This complex can be disrupted by the addition of an excess of unlabeled probes (A and B) in a dose-dependent manner. Preincubation of the UE-1 nuclear protein extracts with antibodies against the EGR-1 prior to the addition of the probe dissociated this complex. Also, this complex was dissociated by addition of an unlabeled Egr-1 consensus sequence (GCE) but not mutated Egr-1 consensus sequence. Probe A also formed a slow-migrating complex with all cells extracts. This complex was not dissociated by EGR-1 antibodies or GCE or unlabeled probe "A" itself, indicating that this complex is not related to EGR-1, possibly a non-specific band. The sums of results demonstrate that EGR-1 can specifically bind to sequences of human FN promoter and activate the transcription of the FN gene.
  • GCE unlabeled Egr-1 consensus sequence
  • EGR-1 -expressing cells Enhanced expression of FN in EGR-1 -expressing cells increases cell adhesion and cell migration.
  • EGR-1 -dependent stimulation of the FN gene produced functional FN in human glioblastoma U251 cells. Because the interaction with extracellular matrix such as FN and PAI-1 is considered to be important for cell adhesion and migration, we tested whether the enhanced expression of FN increased cell adhesion.
  • the EGR-1 -expressing cells (UE-1) and control cells U251 and UN cells were used in attachment efficiency assays. Approximately 28% of EGR-1 -expressing cells UE-1 attached to uncoated plastic plates 3 hours after plating, compared with about 11% for the controls U251 and UN cells (p ⁇ 0.0003).

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Abstract

Cette invention concerne un procédé permettant d'augmenter l'expression d'un facteur anti-métastatique dans une cellule tumorale ou de faire obstacle à la métastase d'une cellule tumorale, faisant intervenir l'administration à une cellule tumorale d'un vecteur comprenant une séquence d'acide nucléique codant pour l'EGR ou une séquence d'acide nucléique codant pour une partie active de l'EGR. La présente invention comprend également: un procédé d'identification de composés ou de compositions permettant d'augmenter l'expression de l'EGR dans une cellule; des procédés d'identification de composés ou de compositions permettant d'augmenter ou de limiter l'expression de l'EGR dans une cellule, l'expression d'au moins un facteur anti-métastatique, ou l'adhérence d'une cellule à un substrat comprenant les compositions pharmaceutiques identifiées grâce à ces procédés; des procédés permettant de faire obstacle à la prolifération d'une cellule tumorale par augmentation de l'expression d'un facteur métastatique.
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