WO1998047526A1 - Modulation de la croissance cellulaire par l'inhibine - Google Patents

Modulation de la croissance cellulaire par l'inhibine Download PDF

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Publication number
WO1998047526A1
WO1998047526A1 PCT/AU1998/000292 AU9800292W WO9847526A1 WO 1998047526 A1 WO1998047526 A1 WO 1998047526A1 AU 9800292 W AU9800292 W AU 9800292W WO 9847526 A1 WO9847526 A1 WO 9847526A1
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Prior art keywords
inhibin
mammal
cells
prostate
cell growth
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PCT/AU1998/000292
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English (en)
Inventor
Gail Petuna Risbridger
David Morritz De Krester
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Monash University
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Publication date
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Priority to AU70151/98A priority Critical patent/AU745971B2/en
Priority to EP98916648A priority patent/EP1011715A4/fr
Publication of WO1998047526A1 publication Critical patent/WO1998047526A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57434—Specifically defined cancers of prostate
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/575—Hormones

Definitions

  • the present invention relates generally to a method of modulating cell growth and more particularly, to a method of modulating prostate cell growth. Even more particularly, the present invention provides a method of treating prostate cancer by inhibiting division of malignant prostate cells.
  • the present invention also relates to a method of screening for a mammal having prostate cancer or a predisposition to prostate cancer.
  • Inhibins are gonadal derived hormones which have a negative feedback action on the release of pituitary follicle stimulating hormone (FSH). They consist of an ⁇ and either a ⁇ A or ⁇ B subunit linked by disulphide bonds (Burger et al. , 1996). Inhibin A is formed by the dimerisation of ⁇ and ⁇ A subunits; inhibin B from dimerisation of ⁇ and ⁇ B subunits. The ⁇ -inhibin subunit is synthesised in precursor forms consisting of pre, pro, ⁇ N and ⁇ C components.
  • FSH pituitary follicle stimulating hormone
  • the precursor ⁇ and ⁇ subunits link to form a 105 kD bioactive inhibin, which forms 31-34 kD inhibin ⁇ C- ⁇ after postranslational modification (cleavage of the pre, pro and ⁇ N regions from the ⁇ -subunit and pro from the ⁇ subunit) (Robertson et al, 1994).
  • Inhibin B is considered to be the physiologically important form of inhibin which regulates FSH release in men (Illingworth et al, 1996). Dimerisation of two ⁇ subunits results in the formation of activin.
  • activin A Activin A
  • Activin B ⁇ B ⁇ B
  • Activin AB ⁇ A ⁇ B
  • the activins stimulate pituitary FSH (Ling et al., 1986).
  • inhibin ⁇ subunits show approximately 30% homology with the ⁇ subunits of TGF ⁇ and thus, inhibins are members of the TGS ⁇ superfamily of growth and differentiation factors (Massague, 1990). In accordance with this classification, the inhibins have been shown to have a wide range of effects, in addition to the regulation of FSH. In erythroid, immune and endocrine tissues, both proliferative and antiproliferative actions of inhibin has been described (Mather et al, 1990; Hedger et al, 1989; Kaipia et al., 1994). Activin A has also been reported to induce apoptosis (Nishihara et al., 1993).
  • inhibins can be antagonised by activins (Hseuh et al., 1987).
  • the actions of activins are mediated through specific serine/threonine kinase receptors (Matthews et al, 1991). No specific receptors for inhibins have been isolated to date.
  • binding proteins for activins which include follistatins (Nakamura et al., 1990).
  • Follistatins have no structural homology to inhibins or activins but can bind strongly to activins and, in doing so, suppress or neutralise their bioactivity (Mather et al, 1993).
  • FS288 Two mRNA species have been identified for follistatin which arise from alternate splicing, and result in two proteins denoted FS288 and FS315.
  • FS288 has been demonstrated to be membrane associated, while FS315 is a secreted protein (Michel et al, 1990).
  • one aspect of the present invention relates to a method of modulating cell growth in a mammal said method comprising administering to said mammal an effective amount of an agent for a time and under conditions sufficient to modulate the expression of a genetic sequence encoding inhibin.
  • inhibin should be read as including reference to all forms of inhibin and fragments thereof or derivatives, homologues, analogues, mutants and variants thereof including all subunit polypeptides thereof including by way of example any protein encoded by the ⁇ or ⁇ subunit gene, the monomeric ⁇ -subunit polypeptide, the subunit precursor polypeptides pre, pro ⁇ N and ⁇ C, the monomeric ⁇ subunit polypeptide, the dimeric ⁇ polypeptide (for example ⁇ A , ⁇ B , ⁇ c , ⁇ D , and ⁇ E ) the dimeric precursor ⁇ C- ⁇ polypeptide and including, but not limited to, derivatives, homologues, analogues, mutants and variants thereof.
  • said inhibin is the ⁇ -subunit polypeptide ( ⁇ -inhibin) or fragment thereof or derivative, homologue, analogue, mutants and variants thereof.
  • ⁇ -inhibin hereinafter, is not intended to be limiting and should be read as including reference to all forms of ⁇ -inhibin including any protein encoded by the ⁇ -subunit gene, all subunit polypeptides thereof including by way of example the monomeric subunit precursor polypeptides pre, pro ⁇ N and ⁇ C, and including, but not limited to, derivatives, homologues, analogues, mutants and variants thereof.
  • the present invention relates to a method of modulating cell growth in a mammal said method comprising administering to said mammal an effective amount of an agent for a time and under conditions sufficient to modulate the expression of a genetic sequence encoding ⁇ -inhibin.
  • mammal includes humans, primates, livestock animals (eg. horses, cattle, sheep, pigs, donkeys), laboratory test animals (eg. mice, rats, rabbits, guinea pigs), companion animals (eg. dogs, cats) and captive wild animals (eg. kangaroos, deer, foxes).
  • livestock animals eg. horses, cattle, sheep, pigs, donkeys
  • laboratory test animals eg. mice, rats, rabbits, guinea pigs
  • companion animals eg. dogs, cats
  • captive wild animals eg. kangaroos, deer, foxes
  • modulating means up-regulating or down-regulating. Accordingly, although the preferred method is to increase the expression of a genetic sequence encoding ⁇ - inhibin, the reduction of the expression of a genetic sequence encoding ⁇ -inhibin expression may also be desired under certain circumstances.
  • expression refers to the synthesis of a polypeptide utilising the mechanisms of transcription and translation of a nucleic acid molecule.
  • modulation of the expression of a genetic sequence encoding ⁇ -inhibin by the administration of an agent to a mammal can be achieved via one of several techniques including but in no way limited to:
  • Said gene may be an ⁇ -inhibin gene or some other gene which directly or indirectly regulates the expression of an ⁇ -inhibin gene.
  • expression of a genetic sequence encoding ⁇ -inhibin expression is modulated in prostate cells and even more preferably the prostate cells are malignant.
  • a method of modulating malignant prostate cell growth in a mammal comprising administering to said mammal an effective amount of agent for a time and under conditions suffient to modulate the expression of a genetic sequence encoding ⁇ -inhibin.
  • the basal epithelial cells of the prostate gland are the predominant site of the expression of the ⁇ -inhibin gene.
  • the synthesis and production of the ⁇ -inhibin subunit protein in prostatic basal epithelium correlates with data demonstrating that the ⁇ -inhibin subunit proteins are localised in these cells. Since both inhibin ⁇ and ⁇ subunits are expressed in the same cells the tissues have the ability to produce ⁇ dimeric inhibin protein.
  • ⁇ -inhibin mRNA and protein is observed in epithelial cells in benign prostate tissues and basal cell hyperplasia but not in poorly differentiated malignant prostate epithelial cells is consistent with tumour suppressive activity of ⁇ -inhibin in the prostate gland.
  • said expression of a genetic sequence encoding ⁇ - inhibin is up-regulated.
  • up-regulation of a genetic sequence encoding ⁇ - inhibin inhibits cell growth.
  • the present invention relates to a method of inhibiting malignant prostate cell growth in a mammal said method comprising administering to said mammal an effective amount of an agent for a time and under conditions sufficient to up-regulate the expression of a genetic sequence encoding ⁇ - inhibin.
  • the modulation of cell growth in a mammal via the modulation of the expression of a genetic sequence encoding inhibin can also be achieved by the administration of inhibin to said mammal.
  • another aspect of the present invention relates to a method of modulating cell growth in a mammal said method comprising administering to said mammal an effective amount of inhibin.
  • said cells are prostate and even more preferably said prostate cells are malignant.
  • the present invention relates to a method of inhibiting malignant prostate cell growth in a mammal said method comprising administering to said mammal an effective amount of inhibin.
  • said inhibin is ⁇ -inhibin. It has been observed that there is a high degree of homology between inhibins from different mammalian species.
  • the inhibin used may be derived from any origin including human, primate, bovine, ovine, porcine or other mammalian or animal species.
  • the inhibin is recombinant human inhibin.
  • inhibitor used herein include fragments, said fragments having the functional activity of inhibin and including but not limited to homologues, analogues, mutants, variants and derivatives thereof. This includes homologues, analogues, mutants, varia and derivatives derived from natural or recombinant sources including fusion proteins. Reference to “inhibin” should also be understood to encompass inhibin agonists.
  • the homologues, analogues, mutants, variants and derivatives may be derived from insertion, deletion or substitution of amino acids in the inhibin.
  • Amino acid insertional derivatives of inhibin used in the present invention include amino and/or carboxylic terminal fusions as well as intra-sequence insertions of single or multiple amino acids.
  • Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in the protein although random insertion is also possible with suitable screening of the resulting product.
  • Deletional variants are characterised by the removal of one or more amino acids from the sequence.
  • Substitutional amino acid variants are those in which at least one residue in the sequence has been removed and a different residue inserted in its place. Additions to amino acid sequences include fusions with other peptides, polypeptides or proteins. Typical substitutions are those made in accordance with Table 1 : TABLE 1 Suitable residues for amino acid substitutions
  • the inhibin of the present invention may be in monomeric or multimeric form meaning that two or more molecules are associated together. Where the same inhibin molecules are associated together, the complex is a homomultimer. An example of a homomultimer is a homodimer. Where at least one inhibin molecule is associated with at least one non- inhibin molecule, then the complex is a heteromultimer such as a heterodimer.
  • Inhibin suitable for use in the present invention may be the inhibin glycoprotein which has a molecular weight of 31 kD in its dimeric form and is made up of a 20 kD ⁇ -subunit and a 14 kD ⁇ -subunit.
  • the inhibin used in the present invention may be that described in Robertson et al., (1985) or Forage et al, or similar.
  • the inhibin suitable for use in the method of treatment aspect of the present invention is not related to the "inhibin" described in WO93/25224 which is a non-glycosylated protein occurring in two forms having molecular weight of 10.5 kD and 16 kD.
  • the preferred method is to down-regulate cell growth, in particular malignant prostate cell growth, the up-regulation of cell growth may be desired under certain circumstances.
  • the present invention provides a method of modulating cell growth in a mammal said method comprising administering a mammalian cell growth modulating effective amount of an inhibin antagonist to said cells.
  • said cells are prostate cells.
  • the present invention provides a method of modulating growth of mammalian prostatic cells comprising administering a prostatic cell growth modulating amount of an inhibin antagonist to said cells.
  • the antagonists may be any compound capable of blocking, inhibiting, or otherwise preventing inhibin from carrying out its normal biological functions in prostate cells or tissue.
  • Antagonists include monoclonal antibodies specific for inhibin, or parts of inhibin, and antisense nucleic acids which prevent transcription or translation of inhibin genes or mRNA in mammalian cells.
  • Antagonists also include analogues of inhibin which bind the inhibin receptors and thereby prevent inhibin from performing its normal biological functions in the prostate.
  • Antagonists in the form of analogues may include those analogues described above.
  • Antisense sequences based on the nucleotide sequences of inhibin disclosed in US Patent 4,740,587 and Forage et al (1986) are also contemplated.
  • agent, inhibin, or inhibin antagonist used may also be linked to a targeting means, such as a monoclonal antibody, which provides specific delivery of the agent, inhibin or antagonist to the cells.
  • a targeting means such as a monoclonal antibody, which provides specific delivery of the agent, inhibin or antagonist to the cells.
  • the agent, inhibin or inhibin antagonist used in the method is linked to an antibody specific for the prostate to enable specific delivery to this organ.
  • Administration of the agent, inhibin, or inhibin antagonists, in the form of a pharmaceutical composition may be performed by any convenient means.
  • the agent, inhibin or inhibin antagonists of the pharmaceutical composition are contemplated to exhibit therapeutic activity when administered in an amount which depends on the particular case. The variation depends, for example, on the human or animal and the inhibin or inhibin antagonist chosen. A broad range of doses may be applicable. Considering a patient, for example, from about 0.1 mg to about 1 mg of inhibin or antagonist may be administered per kilogram of body weight per day. Dosage regimes may be adjusted to provide the optimum therapeutic response.
  • the inhibin or part thereof or antagonist may be administered in a convenient manner such as by the oral, intravenous (where water soluble), intraperitoneal, intramuscular, subcutaneous, intradermal or suppository routes or implanting (e.g. using slow release molecules) .
  • these peptides may be administered in the form of pharmaceutically acceptable nontoxic salts, such as acid addition salts or metal complexes, e.g. with zinc, iron or the like (which are considered as salts for purposes of this application).
  • the tablet may contain a binder such as tragacanth, corn starch or gelatin; a disintegrating agent, such as alginic acid; and a lubricant, such as magnesium stearate.
  • tumour suppressing action of inhibins may be mediated through specific receptor complexes similar to those described for TGF- ⁇ and activin.
  • receptors for inhibins have not been identified although there is a body of indirect evidence to suggest that such receptors exist (Woodruff et al. , 1992; Krummen et al. , 1994).
  • a further aspect of the present invention relates to the use of the invention in relation to human disease conditions.
  • the present invention is particularly useful, but in no way limited to use in inhibiting growth of malignant prostate cells.
  • another aspect of the present invention relates to a method of treating a mammal said method comprising administering to said mammal an effective amount of an agent for a time and under conditions sufficient to modulate the expression of a genetic sequence encoding inhibin.
  • said inhibin is ⁇ -inhibin.
  • said cells are prostate cells and even more preferably said prostate cells are malignant.
  • the present invention relates to a method of treating malignant prostate cells in a mammal said method comprising administering to said mammal an effective amount of an agent for a time and under conditions sufficient to up-regulate the expression of a genetic sequence encoding ⁇ -inhibin.
  • the treatment of a mammal by the administration of an effective amount of an agent for a time and under conditions sufficient to modulate the expression of a genetic sequence encoding inhibin and thereby regulating cell growth can also be achieved by the administration of inhibin to said mammal.
  • another aspect of the present invention relates to a method of treating a mammal said method comprising administering to said mammal an effective amount of inhibin.
  • said inhibin is ⁇ -inhibin.
  • said cells are prostate cells and even more preferably said prostate cells are malignant.
  • the present invention relates to a method of treating malignant prostate cells in a mammal said method comprising administering to said mammal an effective amount of ⁇ -inhibin.
  • the present invention relates to the use of an agent capable of modulating the expression of a genetic sequence encoding inhibin in the manufacture of a medicament for the modulation of cell growth in a mammal.
  • said cells are prostate and even more preferably said prostate cells are malignant.
  • inhibin is ⁇ -inhibin.
  • expression of a genetic sequence encoding ⁇ -inhibin is up-regulated.
  • Yet another aspect of the present invention relates to the use of inhibin in the manufacture of a medicament for the modulation of cell growth in a mammal.
  • said cells are prostate and even more preferably said prostate cells are malignant.
  • inhibin is ⁇ -inhibin.
  • a related aspect of the present invention relates to agents for use in modulating the expression of a genetic sequence encoding inhibin wherein modulating expression of said genetic sequence regulates cell growth.
  • said inhibin is ⁇ -inhibin.
  • said cells are prostate cells and even more preferably said prostate cells are malignant.
  • ⁇ -inhibin is up-regulated. Yet even more preferably cell growth is inhibited.
  • the present invention relates to inhibin for use in regulating cell growth.
  • said inhibin is ⁇ -inhibin.
  • said cells are prostate cells and even more preferably said prostate cells are malignant.
  • the mammal undergoing treatment may be human or an animal in need of therapeutic or prophylactic treatment of a prostate disorder or a potential prostate disorder.
  • tumour suppressive function of ⁇ -inhibin protein is predicated on the observation that ⁇ -inhibin mRNA and protein is present in the prostate basal epithelial cells of patients with benign prostate disease and in said epithelial cells located in non-malignant regions of prostatic tissue from patients exhibiting prostate cancer but not in the malignant regions of said cancerous prostates.
  • another aspect of the present invention relates to a method of screening for a mammal having prostate cancer or a predisposition to prostate cancer, said method comprising screening for the down-regulation of the inhibin protein levels and/or gene expression in said mammal, wherein the down- regulation of the inhibin protein levels and/or gene expression is indicative of said mammal being predisposed to prostate cancer or having already developed prostate cancer.
  • Reference to "down- regulation” should be understood to include reference to the complete absence or total loss of protein and/or gene expression.
  • “Inhibin” has the same meaning as given above and should therefore be understood to include any protein, or fragment thereof, encoded by the ⁇ or ⁇ subunit gene whether existing as a monomer, multimer or fusion protein.
  • Examples of a multimer include the ⁇ heterodimer or a heterodimer comprising any protein encoded by the ⁇ -subunit gene in association with any other protein.
  • said inhibin is ⁇ -inhibin.
  • ⁇ -inhibin also has the same meaning as given above. Accordingly, reference to ⁇ - inhibin includes, by way of example, reference to any protein, or fragment thereof, encoded by the ⁇ -subunit gene, whether existing as a monomer, multimer or fusion protein. Proteins encoded by the ⁇ -subunit gene include, for example, pre-pro- ⁇ N- ⁇ C, pro- ⁇ C and the cleavage products ⁇ N, ⁇ C, pre-pro or isoforms thereof.
  • the ⁇ -inhibin proteins which are detectable in the prostates from patients diagnosed with benign prostate hyperplasia or in the non-malign? t regions of prostate may comprise, for example, ⁇ N and/or ⁇ C regions.
  • the present invention is exemplified, but not limited in any way, by referernce to detection of ⁇ -inhibin levels via the detection of the ⁇ N or ⁇ C regions of the ⁇ -inhibin protein, ⁇ -inhibin proteins comprising ⁇ N and/or ⁇ C regions are also referred to as precursor ⁇ -subunit proteins.
  • the ⁇ N and/or ⁇ C regions of precursor ⁇ - subunit proteins are found to exist either as part of an existing precursor ⁇ -subunit protein or in isolation, for example, following cleavage of said region from a precursor ⁇ -subunit protein.
  • Precursor ⁇ -subunit proteins exist in many forms including, but not limited to, the forms pre- pro- ⁇ N - ⁇ C and pro- ⁇ C.
  • detection of ⁇ -inhibin proteins, including precursor ⁇ -subunit proteins includes the detection of the ⁇ N and/or ⁇ C regions both in isolation, and as part of one or more of the various forms of precursor ⁇ - subunit protein.
  • the ⁇ C and ⁇ N regions can be detected, either in isolation or as part of a precursor ⁇ -subunit protein, using, for example, the polyclonal antibodies # ⁇ C41 and ⁇ N320, respectively.
  • a preferred embodiment of the present invention relates to a method of screening for a mammal having prostate cancer, said method comprising screening for the down-regulation of ⁇ C or isoform thereof in said individual, wherein the down-regulation of the ⁇ C or isoform thereof is indicative of prostate cancer.
  • the present invention relates to a method of screening for a mammal having prostate cancer said method comprising screening for the down-regulation of ⁇ N or isoform thereof in said individual, wherein the down-regulation of ⁇ N or isoform thereof is indicative of prostate cancer.
  • the present invention relates to a method of screening for a mammal having prostate cancer said method comprising screening for the down- regulation of ⁇ -subunit gene expression in said individual, wherein the down- regulation of ⁇ -subunit gene expression is indicative of prostate cancer.
  • a related embodiment of the present invention relates to a method of screening for a mammal having a predisposition to prostate cancer, said method comprising screening for ⁇ -subunit gene expression in said individual, wherein ⁇ -subunit gene expression reveals disruption of the basement membrane, said disruption indicating a predisposition to prostate cancer.
  • the absence of ⁇ -inhibin protein expression in the malignant prostate results in the inability of ⁇ subunit protein monomers to form inhibin ⁇ dimers. Since activin is formed by the dimerisation of two ⁇ subunits, modulation of activin levels in the prostate provides an additional and/or alternative indicator of malignancy.
  • another aspect of the present invention relates to a method of screening for a mammal having prostate cancer or a predisposition to prostate cancer, said method comprising screening for the modulation of activin protein levels in said mammal, wherein the modulation of activin protein levels is indicative of said mammal being predisposed to prostate cancer or having already developed prostate cancer.
  • This method is particularly important for prostate cancer.
  • the mammal is human.
  • the target inhibin molecules in the biological sample are exposed to a specific antibody which may or may not be labelled with a reporter molecule.
  • a bound target may be detectable by direct labelling with an antibody.
  • a second labelled antibody, specific to the first antibody is exposed to the target-first antibody complex to form a target-first antibody-second antibody tertiary complex. The complex is detected by the signal emitted by the reporter molecule.
  • reporter molecule as used in the present specification, is meant a molecule which, by its chemical nature, provides an analytically identifiable signal which allows the detection of antigen-bound antibody. Detection may be either qualitative or quantitative.
  • the most commonly used reporter molecules in this type of assay are either enzymes, fluorophores or radionuclide containing molecules (i.e. radioisotopes) and chemiluminescent molecules.
  • an enzyme is conjugated to the second antibody, generally by means of glutaraldehyde or periodate.
  • glutaraldehyde or periodate As will be readily recognized, however, a wide variety of different conjugation techniques exist, which are readily available to the skilled artisan.
  • Commonly used enzymes include horseradish peroxidase, glucose oxidase, beta-galactosidase and alkaline phosphatase, amongst others.
  • the substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable colour change. Examples of suitable enzymes include alkaline phosphatase and peroxidase.
  • fluorogenic substrates which yield a fluorescent product rather than the chromogenic substrates noted above.
  • the enzyme-labelled antibody is added to the first antibody hapten complex, allowed to bind, and then the excess reagent is washed away. A solution containing the appropriate substrate is then added to the complex of antibody-antigen-antibody. The substrate will react with the enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantitated, usually spectrophotometrically, to give an indication of the amount of hapten which was present in the sample.
  • fluorescent compounds such as fluorescein and rhodamine
  • fluorescein and rhodamine may be chemically coupled to antibodies without altering their binding capacity.
  • the fluorochrome-labelled antibody When activated by illumination with light of a particular wavelength, the fluorochrome-labelled antibody adsorbs the light energy, inducing a state to excitability in the molecule, followed by emission of the light at a characteristic colour visually detectable with a light microscope.
  • the fluorescent labelled antibody is allowed to bind to the first antibody hapten complex. After washing off the unbound reagent, the remaining tertiary complex is then exposed to the light of the appropriate wavelength the fluorescence observed indicates the presence of the hapten of interest.
  • Immunofluorescene and EIA techniques are both very well established in the art and are particularly preferred for the present method. However, other reporter molecules, such as radioisotope, chemiluminescent or bioluminescent molecules, may also be employed.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an agent capable of modulating expression of a genetic sequence encoding inhibin thereby regulating cell growth and one or more pharmaceutically acceptable carriers and/or diluents.
  • said inhibin is ⁇ -inhibin.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an agent capable of regulating expression of a genetic sequence encoding ⁇ -inhibin expression thereby regulating cell growth.
  • expression of a genetic sequence encoding ⁇ - inhibin is up-regulated.
  • up-regulation of expression of a genetic sequence encoding ⁇ -inhibin inhibits cell growth.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an agent capable of up-regulating expression of a genetic sequence encoding ⁇ -inhibin thereby inhibiting cell growth and one or more pharmaceutically acceptable carriers and/or diluents.
  • said cells are prostate cells and even more preferably said prostate cells are malignant.
  • Another aspect of the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising inhibin capable of regulating cell growth and one or more pharmaceutically acceptable carriers and/or diluents.
  • said inhibin is ⁇ -inhibin.
  • ⁇ -inhibin inhibits cell growth.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising ⁇ - inhibin capable of inhibiting cell growth and one or more pharmaceutically acceptable carriers and/or diluents.
  • said cells are prostate cells and even more preferably said prostate cells are malignant.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion or may be in the form of a cream or other form suitable for topical application. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of superfactants.
  • the preventions of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride.
  • Prolonged abso ⁇ tion of the injectable compositions can be brought about by the use in the compositions of agents delaying abso ⁇ tion, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by inco ⁇ orating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilisation.
  • dispersions are prepared by inco ⁇ orating the various sterilised active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.
  • the active ingredients When the active ingredients are suitably protected they may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be inco ⁇ orated directly with the food of the diet.
  • the active compound For oral therapeutic administration, the active compound may be inco ⁇ orated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 1 % by weight of active compound.
  • compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit.
  • the amount of active compound in such therapeutically useful compositions in such that a suitable dosage will be obtained.
  • Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 0.1 ⁇ g and 2000 mg of active compound.
  • the tablets, troches, pills, capsules and the like may also contain the components as listed hereafter: a binder such as gum, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring.
  • a binder such as gum, acacia, corn starch or gelatin
  • excipients such as dicalcium phosphate
  • a disintegrating agent such as corn starch, potato starch, alginic acid and the like
  • a lubricant such as magnesium stearate
  • a sweetening agent such as sucrose, lactose or saccharin
  • a flavouring agent such as peppermint, oil of wintergreen, or
  • tablets, pills, or capsules may be coated with shellac, sugar or both.
  • a syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour.
  • any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed.
  • the active compound(s) may be inco ⁇ orated into sustained-release preparations and formulations. The present invention is further described by the following non-limiting figures and/or examples.
  • Figure 1 is a photographic representation of analysis of ⁇ -subunit mRNA by RT-PCR.
  • Figure 2a is a graphical representation of radioactive profiles of inhibin tracer incubated .- prostate cytosol for 2 days.
  • Figure 2b is a graphical representation of radioactive profile of activin tracer incubated ⁇ prostate cytosol.
  • Figure 3 is a graphical representation of logit plots of B/B 0 recombinant human activin standard (Has3) and rat prostate cytosol.
  • Figure 4 is a photographic representation of the localisation of inhibin ⁇ , ⁇ A and ⁇ B subunit proteins to BPH prostate tissue.
  • A-C Prostate biopsy tissue (A) did not exhibit any inhibin ⁇ immunoreactivity in the glandular epithelium or the stroma. In contrast, using the same ⁇ -subunit antibody, specific immunoreactivity was localised to the stromal cells of a benign mucinous cystadenoma of the ovary (C). Control prostate tissue incubated with anti-mouse IgG did not detect any positive immunoreactivity (B).
  • D-F Prostate biopsy tissues (D:x20 magnification; E:x40 magnification) stained positively using the ⁇ A subunit antibody, and specific immunoreactivity was localised to the glandular epithelium. Note that there was variable staining within the glandular epithelium itself, as indicated by arrows. Control tissue incubated with normal rabbit serum did not show any positive immunoreactivity (F).
  • G-I Prostate biopsy tissue immunostained with the ⁇ B subunit antibody, showed weak immunoreactivity which was localised to the glandular epithelium (G & H). No specific localisation was recorded in control tissue incubated with the anti-mouse IgG antibody (I).
  • Scale bar in A represent 20 microns and is applicable to A, B, C, D, F, G and I.
  • Scale bar in E represent 20 microns and is applicable to E, G and H).
  • Figure 5 is a photographic representation of RT-PCR and Southern analysis of inhibin ⁇ , ⁇ A and ⁇ B subunits, the putative activin ⁇ c subunit, the activin type II receptor (ActRII) and FS288 and FS315 mRNA expression in human BPH biopsy samples.
  • mRNA extracted from two groups of human biopsy samples were analysed by RT-PCR and Southern analysis. Total RNA from adult rat testes (t) and adult rat prostate(p) were used as positive controls, water (w) was used as a negative control.
  • the size of the RT-PCR products was confirmed using pGEM DNA molecular weight markers (Promega Biotec, Madison, USA) (m).
  • Figure 6 is a photographic representation of the localisation of inhibin ⁇ C and ⁇ N subunit proteins and ⁇ mRNA to benign prostatic hype ⁇ lasia tissue.
  • the basal cells in the prostatic epithelium of the benign prostate biopsy tissue stained positively using the cytokeratin market antibody (A).
  • Control prostate tissue incubated with mouse IgG did not detect any specific localisation (B).
  • Specific immunoreactivity for inhibin ⁇ C protein was detected in basal cells of the prostate epithelium (C).
  • Control tissue incubated with sheep IgG did not show any positive immunoreactivity (D).
  • Both basal cells and secretory epithelium displayed immunoreactivity for inhibin ⁇ N protein (E). No specific localisation was recorded in the control tissue incubated with sheep IgG (F).
  • ⁇ -inhibin mRNA was expressed in epithelial basal cells in the benign prostate (G) and in one patient, in both basal and secretory epithelial cells (H - note the section has been counterstained) .
  • the localisation was detected with the sense probe (I and J).
  • Figure 7 is a photographic representation of the localisation of inhibin ⁇ C and ⁇ N subunit proteins and ⁇ mRNA to patients with basal cell hype ⁇ lasia. Cytokeratin specific antibody identified areas of basal cell hype ⁇ lasia in benign prostate tissue (A). Incubation of the control section with mouse IgG showed no specific immunoreactivity (B). The same regions displayed positive immunoreactivity for both ⁇ C and ⁇ N inhibin protein (C and E, respectively). Control sections incubated with sheep IgG displayed no positive localisation (D and F, respectively), ⁇ -inhibin mRNA was positively expressed in basal cell hype ⁇ lasia (G). No specific localisation was detected with the sense probe (H).
  • Figure 8 is a photographic representation of the localisation of inhibin ⁇ C and ⁇ N subunit proteins and ⁇ -inhibin mRNA to non-malignant and malignant regions of prostate tissue from patients with high grade prostate cancer.
  • Inhibin ⁇ C protein was localised to the basal epithelial cells in the non-malignant region (A) of the prostate biopsy.
  • the adjacent tumour cells displayed no positive immunoreactivity (B).
  • Specific localisation of ⁇ N protein was observed in the secretory epithelium of the non-malignant region (C); the adjacent tumour tissue displayed no positive staining (D).
  • a control section was incubated with sheep IgG and displayed no specific immunoreactivity (E and F).
  • ⁇ -inhibin mRNA was expressed in basal epithelial cells in the non-malignant region (G).
  • the adjacent malignant region showed no positive localisation (H).
  • the control section incubated with the sense probe displayed no staining (I and J).
  • RNA sequences for the reverse transcription-polymerase chain reaction were taken from van den Eiijnden-van Raaij et al (1992 Dev biol 154: 356-365) and were as follows.
  • the sequence of the downstream primer for inhibin ⁇ was 5' AGC CCA GCT CCT GGA AGG AGA T 3' [SEQ ID NO.1] and the upstream primer was 5' TCA GCC CAG CTG TGG TTC CAC A 3' [SEQ ID NO: 2].
  • an intron is absent.
  • Predicted fragment sizes were ⁇ -subunit 444bp.
  • the oligonucleotide primers for the activin receptors type II and IEB were designed from the rat sequence data available on Genebank: the sequence for the downstream primer of ActR- II was 5' GGA ATT CGC ACC AAT GAA CTG 3' [SEQ ID NO.3] and the upstream primer was 5' CGG GAT CCA ACT GCT ATG ACA GG 3' [SEQ ID NO:4].
  • the internal primer used for the southern detection was as follows 5' TAG GAC AAT GTG GCT TCG GGT GG 3' [SEQ ID NO: 5]. These primers span the extracellular ?.nd transmembrane regions of the gene and the predicted fragment size is 510 bp.
  • the ActR-IIb primers are as follows, downstream 5' AGC CAG CAC CGC GGT GAG 3' [SEQ ID NO:6] and upstream 5' GTG GCT GTG AAG ATC TCC 3' [SEQ ED NO:7].
  • the internal primer used for the southern analysis is as follows 5' TGG CTC ATC ACA GCC TT 3' [SEQ ID NO:8]. These primers span the serine kinase domain and the predicted product size is 366 bp.
  • Reverse transcription Reverse transcription was carried out using 0.5 ⁇ g total RNA mixed with 4U of AMV reverse transcriptase (Promega Biotec, Madison, WT), 20U of RNasin (Promega Biotec, Madison, WI) ImM dNTP, lmM MgCl 2 , 25pmol of the appropriate downstream primer in PCR buffer (Biotec International, Ltd, WA, Australia) to a final volume of 20 ⁇ l. The solution was incubated at 42°C for 2 hours, then heated to 95 °C for 5 min, and cooled rapidly on ice.
  • the PCR was performed in an automatic DNA thermal cycler (Corbett Research Mort Lake Australia) as previously described by Saiki et al, (1988 Science 239: 487-491). Briefly 5 ⁇ l of the RT mixture was added to 0.2mM dNTP, 25pmol of the appropriate upstream primer and 2U of the thermostable Tth DNA polymerase (Biotec International Ltd, WA, Australia) in the PCR buffer (Biotec International Ltd, WA, Australia) in a final volume of 20 ⁇ l. Denaturation was at 95 °C for 30 sec, annealing at 56°C for ⁇ -subunit, 55°C Act RII and 46 °C for ActRIIB for 30 sec, and extension at 72 °C for 1 min for a 40 cycle program. The products were then analysed by agarose gel electrophoresis in IX TAE.
  • This Example indicates that activin and inhibin are present in the prostate, and that activin is produced by the prostate itself.
  • Phenylmethyl sulfonyl fluoride (PMSF), BSA, and Triton were purchased from Sigma (St Louis MO).
  • Deoxycholate, Tween 20, Sodium chloride and EDTA were purchased from BDH/Merck (Australia). SDS was from Biorad.
  • Detection af activin degradation In order to determine if residual protease activity in the prostate cytosols was able to degrade activin tracer, aliquots of tracer were incubated at 4°C for 48h in the presence of prostate cytosol or buffer. The incubates were diluted in nonreducing buffer, boiled for 1 min, microfuged for 30s and applied to 15% SDS-acrylamide gels. The resulting gel was sliced into 1mm fractions and the radioactivity measured; the radioactive profiles for buffer controls were compared to those with prostate cytosol and shown to be identical.
  • human recombinant activin A pool G was iodinated by chloramine T and purified by gel filtration and dye affinity chromatography and used in 0.5% BSA + 0.1% Triton in PBS.
  • Antiserum an ovine antiserum which has been previously used for radioimmunoassay was raised to a recombinant B A subunit fusion protein and human recombinant activin A and used at a final dilution of 1 : 120 000 in CTS reagent (0.125M deoxychloate, 5% Tween 20 and 4% SDS). Limited cross reaction of the antiserum with bovine 31 KDa inhibin and human recombinant 34KDa inhibin, TGFBl, MIS and follistatin ⁇ 3.3% has been previously reported (Robertson et al 1992).
  • a method was devised using delayed tracer addition conditions over 4 days at 4°C.
  • a donkey anti-sheep serum (PBS + 0.5% BSA and 0.01M EDTA) was used to separate bound from free activin tracer.
  • the prostate cytosol contained detectable levels of immunoactive activin, which diluted in linear and parallel to that of an activin standard - human recombinant standard 3 (Has3). ( Figure 3). Inhibin immuno activity was also detected in these examples.
  • the levels of activin in prostate cytosols obtained after EDS treatment are shown in Table 2.
  • the data are recorded as ng/organ and ng/g prostate tissue. There is a significant decrease in activin in the prostate and per unit mass of tissue, indicating that activin levels are responsive to androgen withdrawal. The implication of the changes in concentration of activin may have yet to be determined.
  • inhibin RIA detects the pro ⁇ c fragment of inhibin ⁇ -subunit, the RIA may be measuring an alteration in immunoactive inhibin forms which are responsive to androgens.
  • Tissues for immunolocalisation were obtained from archival needle biopsy material from 14 men who received no androgen therapy. At least five sections from each biopsy were used for immunochemistry, with antibodies of defined specificity as described below. Benign prostatic hype ⁇ lasia was confirmed by histologic examination conducted at Melbourne Pathology (Melbourne, Australia).
  • prostate needle biopsy tissues were collected at surgery under sterile conditions. The specimens were wrapped in sterile foil and snap frozen in liquid nitrogen, before storage at -70° C. Pathological examination of tissues taken at surgery, confirmed benign prostatic hype ⁇ lasia. Prostate tissues were obtained from a total of 28 patients, which were grouped according to diagnosis into three groups with BPH, basal cell hype ⁇ lasia or prostate cancer.
  • Needle biopsies were obtained from 16 patients with BPH, 2 patients with basal cell hype ⁇ lasia and 12 patients with prostate cancer (each having a Gleason score grading between seven and ten). None of the patients had received any form of androgen therapy. Two patients with basal cell hype ⁇ lasia were identified by histological examinations and diagnosis. The tissues were fixed in 10% buffered formalin and processed in paraffin.
  • AS #64 Antibody (AS #64) was raised against a human ⁇ A subunit fusion protein and human recombinant activin A in sheep. This antibody has been used for the radioimmunoassay of activin A and has no cross reactivity with Mullerian Inhibiting Substance, TGF ⁇ and ⁇ 3.3% cross reactivity with human recombinant inhibin A (Robertson et al., 1992).
  • the radioimmunoassay using AS #64 has been previously used to purify dimeric activin A to homogeneity from ovine amniotic fluid (de Kretser et al, 1994) and for the detection of activin A in biological fluids and samples (McFarlane et al., 1996).
  • This antibody detects both the monomeric and dimeric forms of activin A, and the cross reactivity of the AS #64 with monomeric ⁇ A in the radioimmunoassay was estimated to be 17% (Robertson et al., 1992).
  • non-immune serum was used as a control, or the antiserum was preabsorbed with human recombinant activin ⁇
  • Preabso ⁇ tion of the antisera was achieved by incubating 5 ⁇ l of undiluted antisera with 1 ⁇ g antigen, either human recombinant activin A or inhibin A, in PBS (200 ⁇ l) at 4°C overnight. The mixture was diluted to a total volume of 1 ml with PBS and centrifuged at 12,000 ⁇ m, and the supernatant decanted and used accordingly.
  • a polyclonal rabbit antibody to inhibin ⁇ A was obtained from Dr W. Vale, of the Salk Institute.
  • a mouse monoclonal antibody to ⁇ B subunit was kindly provided by Dr J. Mather, Genentech (San Francisco, USA). Both have been previously used in the detection of ⁇ subunit proteins in ovarian tumour tissue (Gurusinghe et al., 1995).
  • the follistatin antisera AS #202, was raised in an intact adult male New Zealand rabbit to purified bovine 39 kDa follistatin, and showed ⁇ 0.5 % cross reactivity to bovine inhibin A and bovine activin A (Klein et al, 1991).
  • An antibody to smooth muscle actin was purchased from Dako Co ⁇ oration.
  • the polyclonal antibody # ⁇ 41 was produced by immunisation against recombinant bovine ⁇ C inhibin subunit fusion protein, the sheep was boosted with human ⁇ C inhibin subunit fusion protein and human recombinant inhibin This antibody was used for the detection of the ⁇ C inhibin subunit and has been used previously to measure ⁇ -inhibin levels in serum from normal and postmenopausal women using immunofluorometric assay.
  • a polyclonal antibody # ⁇ 320 was directed to a fragment (amino acid 1-26) of the fusion protein bovine ⁇ N subunit and used to detect the ⁇ N subunit. Immuno staining for cytokeratin was performed using the monoclonal antibody NCL-LP34 obtained from Novacastra Laboratories (Newcastle Upon Tyne, UK).
  • human prostate sections were rehydrated and placed in antigen retrieval solution (Dako, CA, USA) for 20 minutes in a water bath at 85°C. The slides were then washed in PBS and preincubated in CAS block for 30 minutes.
  • Activin A was immunolocalised using AS #64 at a dilution of 1 :200, or ⁇ A monoclonal (1 : 100) and incubated overnight at room temperature.
  • Activin B was localised using the ⁇ B monoclonal antibody (1 : 100), as was follistatin , using the AS #202 (1 : 100).
  • Controls were incubated with antiserum preabsorbed with human recombinant activin A (l ⁇ g/ml), or a mixture of bovine follistatins (35-, 39-, and 45 kDa) purified from follicular fluid (l ⁇ g/ml) or with normal rabbit serum. After overnight incubation, the sections were washed in PBS and incubated with biotinylated rabbit antisheep IgG (activin A), sheep antirabbit ( ⁇ A subunit), rabbit antimouse IgG (monoclonal ⁇ B ) or biotinylated goat antirabbit sera (follistatin) (Vector Laboratories, California, USA, 1:200) for one hour.
  • human recombinant activin A l ⁇ g/ml
  • bovine follistatins 35-, 39-, and 45 kDa
  • Actin staining was localised using the actin antibody (1:50) for 1 hour. Sections were washed 3 times with PBS (0.01M phosphate buffered phosphate; pH 7.4) and then incubated with rabbit anti-mouse IgG (1:200) for 1 hour. After 2 washes in Tris buffer (0.1M Tris-HCl : pH 8), the sections were incubated in Extravidin Alkaline Phosphatase (Sigma, St Louis, MO, USA) (1 : 100) for one hour. The New Fuchsin Substrate Kit (Biogenex, CA, USA) was used for the demonstration of Alkaline phosphatase.
  • Sections were dewaxed, rehydrated and placed in Target Retrieval solution (Dako, Ca ⁇ interia, CA); antigenic sites were exposed by heating at 70°C for 7 minutes. After washing in 0.01M phosphate buffered saline (PBS; 10 mM PO 4 , 154 mM NaCI, pH 7.4), endogenous peroxidase was blocked by 3% H x O 2 for 30 minutes. Sections were incubated with 0.2% Triton X-100 (Sigma Chemical Co. , St. Louis, MO) for 10 minutes and tiV , blocked with 1 : 1 mixture of CAS block (Zymed, San Francisco, CA) and 10% normal rabbit serum at room temperature for 20 minutes.
  • Target Retrieval solution Dako, Ca ⁇ interia, CA
  • Inhibin was localised using the ⁇ C polyclonal antibody (1.6 ⁇ g/ml) and the ⁇ N polyclonal antibody 1.9 ⁇ g/ml). Basal cells were localised using cytokeratin monoclonal antibody (1 : 100). All antibodies were incubated at 4°C overnight. Controls were incubated with sheep (inhibin) or mouse (cytokeratin) IgG at matched dilution or protein or protein concentration.
  • Poly A+ RNA was extracted directly from the tissues using the DynabeadsTM protocol (Dynal, Oslo, Norway). The poly A+ RNA was eluted in 50 ⁇ l of sterile DEPC-treated water and stored at -20 °C until used.
  • Oligonucleotide primers for the ⁇ , ⁇ A and ⁇ B subunits were designed from human cDNA sequence data obtained from Genbank (acces. #M32755 [27], #X57578 [28] and #M 13437 [29]). The oligonucleotide primers were designed to span the single intron, and yield products of 169bp, 336bp and 500bp respectively. ⁇ c primers (Schmitt et al, 1996) are believed to span an intron, based on the homology between the ⁇ A and ⁇ B subunit members, and yield a product of 290bp.
  • PCR primers used for detecting ActRII were designed from the mouse sequence data (Mathews et al., 1991) and span the extracellular and transmembrane domains, to yield a product of 510bp.
  • Follistatin primers (Meinhardt et al.) were designed to span exons 5 and 6 of the human follistatin sequence and yield two products of 207bp and 470bp corresponding to FS 315 and FS 288, respectively.
  • the deduced precursor sequences bear no homology with the ⁇ , ⁇ A and ⁇ B chains.
  • RT Reverse Transcription Reverse transcription for all mRNAs was carried out using 20 ⁇ l of A+ RNA, denatured at 65 °C for 5 min, and mixed with 30 U reverse transcriptase (Promega Biotec, Madison, WI, USA), 40 U RNAsin (Promega Biotec, Madison, WI, USA), 15 pmol Oligo (dT) 15 primer (Promega Biotec, Madison, WI, USA), 1 mM of each dATP, dTTP, dCTP, dGTP (Promega Biotec, Madison, WI, USA) to a final volume of 50 ⁇ l. The solution was incubated at 42°C for 2 hours, heated to 95°C for 2 min and cooled rapidly on ice.
  • PCR was performed in a Perkin Elmer Cetus DNA Thermal Cycler as previously described by Saiki et al, (Saiki et al, 1985). Briefly, 10 ⁇ l of the RT mixture was added to 30 pmol of each primer, and 1 U of Ampli Taq DNA polymerase (Roche Molecular Systems Inc. , Branchburg, New Jersey), in 1 x PCR buffer (Roche Molecular Systems Inc. , Branchburg, New Jersey) to a final volume of 50 ⁇ l. PCR products were analysed by Nusieve GTG agarose gel electrophoresis in 1 x TAE (0.04 M Tris-acetate, 0.001 M EDTA).
  • Probes were labelled either with DIG or with 32 [P] for Southern analyses. Probes for ActRII, ⁇ A and ⁇ c were derived from sequenced PCR products, and were labelled with DIG. Briefly, 25 ng of denatured probe was mixed with 1 x hexanucleotide mix (Boehringer Mannheim GmbH Biochemica, Germany),. 1 x dNTP labelling mix (Boehringer Mannheim GmbH Biochemica, Germany), 2 ⁇ l 0.25 mM DIG-dUTP (pH 6.5), and 5U of Klenow enzyme (Promega Biotec, Madison, WI, USA) to a final volume of 20 ⁇ l. The mixture was incubated at 37 °C for 1 hour. 20mM of EDTA was added to stop the reaction, before storing the probe at -20 °C.
  • the probe was precipitated after the addition of 3 ⁇ g herring sperm DNA (Promega Biotec, Madison, WI, USA), 0.0 M sodium perchlorate, 0.4 vol isopropanol in a final volume of 165 ⁇ l and centrifuged at 13,000g for 5 min. Labelling efficiency was determined by scintillation counting (1900 TR Liquid Scintillation Analyser, Packard Instrument Co. , Ulgersmaweg, The Netherlands).
  • Hybridisation was carried out for 2 hrs at 65°C, before washing for 15 min, twice with 2 x SSC + 0.1 % SDS, and twice 0.5 x SSC + 0.1 % DIG labelled probes were detected using anti-DIG antibody conjugated to alkaline phosphatase, followed colorimetric analysis as per manufacturers directions.
  • 32 [P] labelled probes were detected by auto radiography using XOMAT AR film (Eastman Kodak Co. , NY, USA) with intensifying screens at -75°C.
  • Digoxigenin (Dig) labelled riboprobes were prepared using the method outlined in the Boehringer Mannheim riboprobe labelling kit. Rat and human inhibin ⁇ -subunit share an 82% homology and riboprobes to both rat and human sequences were used in this study.
  • Dig antisense and sense cRNA probes (gift from Dr. Moira O'Bryan, Institute of Reproduction and Development, Monash University, Melbourne, Australia) were synthesised from a ⁇ 400bp partial rat ⁇ -inhibin subunit cDNA cloned into pGem 4Z (Esch et al, 1987). Antisense probes were transcribed from Ec ⁇ RI linearised plasmids with T7 RNA polymerase and sense cRNA was generated from Hindill linearised plasmids with SP6 RNA polymerase. The amount of Dig-labelled RNA was determined by comparison to a Dig-labelled RNA control using dot blot analysis.
  • sections were washed in lxPBS (2x5min) and treated with proteinase K (20 ⁇ g/ml) for 30 min at 37 °C. Following digestion sections were washed in PBS containing 0.2% glycine for 5 min followed by 5 min fixation in 4% Paraformaldeyde. Sections were then washed in PBS 2x5 min, equilibrated for 2 min in 0.1M triethanolamine and acetylated in 0.25% acetic anhydride in Triethanolamine for 5 min.
  • Riboprobe was diluted in hybridisation buffer to a concentration of 200-1000 ⁇ g/ml and denatured at 65 °C for 10 min to remove secondary structures. Slides were then incubated at 80°C for 10 min and hybridisation was performed under coverslips in a humidified box at 42° C overnight.
  • FIG 4 The pattern of localisation of inhibin ⁇ , ⁇ A , and ⁇ B subunits is shown in Figure 4, and was determined using specific monoclonal and polyclonal antibodies to the ⁇ and ⁇ subunit proteins. As shown in Figure 4 A and B, no ⁇ immunoreactivity could be detected in BPH tissues, although ⁇ immunoreactivity was readily detectable in positive control sections of human ovarian benign cystadenoma (Fig 4C). ⁇ A subunit reactivity was predominantly localised to the epithelial tissues (Fig 4D) and it was noted that the staining intensity was variable within, and between, the glandular structures in the same sections (Fig 4D, E). No immunoreactivity was present in the control sections (Fig 4F).
  • RNA samples a-e were used to determine the presence of the activin receptor, ActRII, inhibin ⁇ A and the putative activin ⁇ c mRNA; patent samples f-j were used to determine follistatin, and inhibin ⁇ and ⁇ B subunit mRNA expression.
  • Total RNA from rat testes ( Figure 5, Lane t) and rat prostate ( Figure 5, Lane p) were used as positive controls for each of the primer pairs.
  • Figure 5A-C demonstrate the mRNA for the activin receptor (ActRII), inhibin ⁇ A subunit, and the putative ⁇ c subunit are expressed in human prostate tissue samples a-e.
  • Figure 5A shows the detection of ActRII mRNA in all five biopsy samples from patients a-e (Lanes a-e respectively), and demonstrated the integrity of the extracted mRNA.
  • Inhibin ⁇ A subunit mRNA expression was detected by Southern analysis in three of the five patient samples ( Figure 5B, lanes c, d, e), suggesting variability in ⁇ A mRNA expression. Whereas the putative ⁇ c subunit mRNA was detected in all of the patent samples ( Figure 5C, Lanes a-e).
  • Tissue sections obtained from two patients with basal cell hype ⁇ lasia were used to detect inhibin ⁇ -subunit gene expression and protein localisation was determined. Identification of regions of basal cell hype ⁇ lasia was confirmed using a cytokeratin antibody as shown in Figure 7A. No immunoreactivity was localised in the control section ( Figure 7B). ⁇ C and ⁇ N inhibin subunit protein immunoreactivity was also localised to these regions of the tissue sections and confirmed that inhibin proteins are localised to basal cells as shown in Figure 7C and E, respectively. No immunoreactivity was detected in the control sections ( Figure 7D and F).
  • Table 2 The effect of EDS, 3 and 14 days after administration, on prostate weight and the levels of ir-inhibin and ir-activin in the prostate, expressed as ng/g tissue and ng/organ. Values are means ⁇ S.D., n-5
  • MOLECULE TYPE Oligonucleotide DNA
  • xi SEQUENCE DESCRIPTION: SEQ ID NO : 1 : AGCCCAGCTC CTGGAAGGAG AT 22
  • MOLECULE TYPE Oligonucleotide DNA
  • Xl SEQUENCE DESCRIPTION: SEQ ID NO : 2 : TCAGCCCAGC TGTGGTTCCA C 21
  • MOLECULE TYPE Oligonucleotide DNA
  • xi SEQUENCE DESCRIPTION: SEQ ID NO : 3 : GGAATTCGCA CCAATGAACT G 21
  • MOLECULE TYPE Oligonucleotide DNA
  • xi SEQUENCE DESCRIPTION: SEQ ID NO : 4 : CGGGATCCAA CTGCTATGAC AGG 23 (2) INFORMATION FOR SEQ ID NO .5 •
  • MOLECULE TYPE Oligonucleotide DNA
  • xi SEQUENCE DESCRIPTION: SEQ ID NO : 5 : TAGGACAATG TGGCTTCGGG TGG 23
  • MOLECULE TYPE Oligonucleotide DNA
  • SEQUENCE DESCRIPTION SEQ ID NO : 6 : AGCCAGCACC GCGGTGAG 18
  • MOLECULE TYPE Oligonucleotide DNA
  • xi SEQUENCE DESCRIPTION: SEQ ID NO : 7 : GTGGCTGTGA AGATCTCC 18
  • MOLECULE TYPE Oligonucleotide DNA (XI ) SEQUENCE DESCRIPTION: SEQ ID NO: 9: CGGGATCCAA CTGCTATGAC AG 22
  • MOLECULE TYPE Oligonucleotide DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: GGAATTCGCA CCAARGAACT G 21
  • MOLECULE TYPE Oligonucleotide DNA
  • xi SEQUENCE DESCRIPTION: SEQ ID NO.11: TTCCCTCTGT GATGAGCTGT G 21
  • MOLECULE TYPE Oligonucleotide DNA
  • MOLECULE TYPE Oligonucleotide DNA
  • xi SEQUENCE DESCRIPTION: SEQ ID NO: 13.
  • MOLECULE TYPE Oligonucleotide DNA (xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 14: GTGGCTGCGT ATGTGTTGGG ATG 23
  • MOLECULE TYPE Oligonucleotide DNA
  • xi SEQUENCE DESCRIPTION: SEQ ID NO: 16: AAGAAGATGG TGACTTTGGT C 21 (2) INFORMATION FOR SEQ ID NO: 17:
  • MOLECULE TYPE Oligonucleotide DNA
  • xi SEQUENCE DESCRIPTION: SEQ ID NO: 17: GAAATCATCA GCTTCGCCGA GAC 23
  • MOLECULE TYPE Oligonucleotide DNA
  • xi SEQUENCE DESCRIPTION: SEQ ID NO: 18: GAACTGTTGC CTGCAACAGA GGTTG 25
  • MOLECULE TYPE Oligonucleotide DNA
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:20: TTCACATTCC AGTTCCCTGT TGTC 24 BIBLIOGRAPHY

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Abstract

La présente invention concerne un procédé permettant de moduler la croissance cellulaire, et plus particulièrement, un procédé permettant de moduler la croissance cellulaire de la prostate par l'administration d'inhibine, d'un antagoniste de l'inhibine ou d'un agent qui module l'expression de l'inhibine. Plus particulièrement, l'invention traite d'un procédé de traitement du cancer de la prostate consistant à inhiber la division des cellules malignes de la prostate. La présente invention traite aussi d'un procédé permettant de détecter le cancer de la prostate ou la prédisposition au cancer de la prostate chez le mammifère.
PCT/AU1998/000292 1997-04-23 1998-04-23 Modulation de la croissance cellulaire par l'inhibine WO1998047526A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU70151/98A AU745971B2 (en) 1997-04-23 1998-04-23 Inhibin modulation of cell growth
EP98916648A EP1011715A4 (fr) 1997-04-23 1998-04-23 Modulation de la croissance cellulaire par l'inhibine

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AUPO6388A AUPO638897A0 (en) 1997-04-23 1997-04-23 Modulation of cell growth and methods relating thereto
AUPO6388 1997-04-23

Publications (1)

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WO1998047526A1 true WO1998047526A1 (fr) 1998-10-29

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AU (1) AUPO638897A0 (fr)
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WO2004086048A1 (fr) * 2003-03-28 2004-10-07 Prostate Diagnostics Pty. Ltd. Procede et agents diagnostiques et therapeutiques
WO2005083438A1 (fr) * 2004-02-27 2005-09-09 Monash University Procede de determination d'un pronostic pour des patients atteints d'un cancer a degre modere

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US6051326A (en) * 1997-04-26 2000-04-18 Cabot Corporation Valve metal compositions and method

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004086048A1 (fr) * 2003-03-28 2004-10-07 Prostate Diagnostics Pty. Ltd. Procede et agents diagnostiques et therapeutiques
WO2005083438A1 (fr) * 2004-02-27 2005-09-09 Monash University Procede de determination d'un pronostic pour des patients atteints d'un cancer a degre modere

Also Published As

Publication number Publication date
EP1011715A1 (fr) 2000-06-28
AUPO638897A0 (en) 1997-05-22
EP1011715A4 (fr) 2003-09-24

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