EP1112356A1 - Inhibiteur du brn-3b et son utilisation dans le traitement contre le cancer du sein et le cancer des ovaires - Google Patents

Inhibiteur du brn-3b et son utilisation dans le traitement contre le cancer du sein et le cancer des ovaires

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Publication number
EP1112356A1
EP1112356A1 EP99946334A EP99946334A EP1112356A1 EP 1112356 A1 EP1112356 A1 EP 1112356A1 EP 99946334 A EP99946334 A EP 99946334A EP 99946334 A EP99946334 A EP 99946334A EP 1112356 A1 EP1112356 A1 EP 1112356A1
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EP
European Patent Office
Prior art keywords
brn
inhibitor
expression
substance
vector
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99946334A
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German (de)
English (en)
Inventor
David S. UCL Med. Sc. Div. Path. LATCHMAN
Vishwanie UCL Med. Sc. Div. Pat. BUDHRAM-MAHADEO
Daniel UCL Med. School Div. of Path. NDISANG
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University College London
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University College London
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Publication of EP1112356A1 publication Critical patent/EP1112356A1/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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • This invention relates to inhibitors of Brn-3b expression and/or activity and screening methods for the identification of such inhibitors. It further relates to the use of said 5 inhibitors in the treatment of cancer.
  • the POU (Pit-Oct-Unc) family of transcription factors was originally defined on the basis of a common 150-160 amino acid domain which was found in found in the mammalian transcription factor Pit- 1 , Oct-1 and Oct-2, and the nematode regulatory
  • the common POU domain constitutes the DNA binding domain of these proteins and consists of two portions, a POU-specific domain which is unique to the POU factors and a POU-homeodomain which is related to the homeobox found in a number of other transcription factors.
  • Brn-3a, Brn-3b and Bm-3c are closely related members of the POU family which are encoded by different genes (7) and are expressed in distinct but overlapping patterns in the
  • Brn-3a has been shown to be over-expressed in aggressive neuroendocrine tumours (14), whilst both Brn-3a and Brn-3b are expressed at high levels in human neuroblastomas (15, 16) Brn-3a and Brn-3b have previously been detected in the human breast cancer cell line MCF-7 (13)
  • CLN3 cervical intra-epithelial neoplasia grade 3
  • the present invention is based on the finding that mammary tumour tissue which has reduced expression levels of the BRCA-1 gene shows elevated expression of the Brn-3b POU family transc ⁇ ption factor
  • the BRCA-1 gene was identified on the basis of its mutation in a number of cases of familial breast cancer indicating that its mactivation can cause this disease Athough BRCA-1 does not appear to be mutated in cases of sporadic breast cancer, its expression has been shown to be reduced in a number of cases
  • Brn-3b The elevated expression of Brn-3b is not found in normal mammary cells, benign tumours or malignant tumour samples which do not exhibit reduced levels of the BRCA-1 gene In contrast no correlation between the level of BRCA-1 expression and the expression of the related POU family transcription factor Brn-3a Moreover, Brn-3b but not Brn-3a can strongly repress the BRCA-1 promoter approximately 20 fold in transfections carried out in mammary tumour cells
  • Brn-3b may play an important role m regulating expression of BRCA-1 in mammary tumours with enhanced expression of Brn-3b resulting in reduced BRCA-1 expression and thereby being potentially involved in tumour development
  • the repression of Brn-3b expression by either pharmacological or by gene therapy procedures represents a potential method for treating breast cancers
  • the invention provides an inhibitor of Brn-3b expression and/or activity for use m a method of treatment of the human of animal body
  • Such inhibitors are useful, in particular, in the treatment of breast cancer and/or ova ⁇ an cancer
  • the invention also provides a method for identifying an inhibitor of Brn-3b expression comprising (a) providing a test construct comprising a Brn-3b promoter operably linked to a coding sequence, (b) contacting a substance to be tested with the test construct under conditions that would permit the polypeptide encoded by the said coding sequence to be expressed in the absence of the said substance; and
  • the invention further provides a pharmaceutical composition comprising an inhibitor of Brn-3b expression and/or activity and a therapeutically acceptable carrier or diluent.
  • the inhibitors of Brn-3b expression and/or activity may be used in treating breast or ovarian cancer and therefore the invention provides a method of treatment of treating a host suffering from breast cancer or ovarian cancer, which method comprises administering to the host a therapeutically effective amount of an inhibitor of Bra-3a expression and/or activity.
  • Figure 1 shows levels of Brn-3a mRNA as determined by RT/PCR assay in normal/b enign mammary material or in malignant mammary tumours from pre- menopausal or post-menopausal women. Bar designates the mean with standard deviation
  • Figure 2 shows levels of Brn-3b mRNA as determined by RT/PCR assay in normal benign mammary material or in malignant mammary tumours from pre- menopausal or post-menopausal women. Bar designates the mean with standard deviation (SD) and population size 'n' .
  • SD standard deviation
  • Figure 3 shows comparison of the mRNA levels of Brn-3b and BRCA-1 in malignant mammary tumour material obtained from pre (panel a) or post (panel b) menopausal women or from benign mammary material (panel c). Brn-3b levels are shown as solid bars, BRCA-1 levels as open bars.
  • Figure 4 shows comparison of the mRNA levels of Bm-3a and BRCA-1 in malignant mammary tumour material obtained from pre (panel a) or post (panel b) menopausal women or from benign mammary material (panel c) Brn-3a levels are shown as solid bars, BRCA-1 levels as open bars
  • Figure 5 shows comparison of BRCA-1 levels in mammary tumours with low or high levels of Brn-3b Bar designates the mean with standard deviation (SD) and population size 'n'
  • Figure 6 shows a luciferase reporter assay of MCF7 cells co-transfected with a promoter/reporter construct containing the full length BRCA-1 promoter together with expression vector lacking any insert (V) or the same vector expressing either Brn-3a or Brn-3b Values have been equalized relative to the activity obtained in the co-transfection with the reporter construct and empty expression vector (set at 100%) and are the mean of five determinations whose standard deviation is shown by the bars
  • any suitable inhibitor of Brn-3b expression or activity may be employed in the present invention
  • the expression of Brn-3b in a cell may be reduced by presence in that cell of a polynucleotide which can hybridize to the Brn-3b mRNA Therefore a polynucleotide which is capable of hyb ⁇ dizing to Brn-3b mRNA can constitute an appropriate inhibitor of Brn-3b expression
  • two approaches are as follows
  • Antisense RNA The delivery of a nucleic acid construct which allows the expression of an RNA which can hybridize to the Brn-3b mRNA This results in the formation of an RNA-RNA duplex which may result in the direct inhibition of translation and/or the destabihzation of the target message, for example, rendering susceptibility to nucleases Therefore, the nucleic acid construct will typically lead to the expression of a polynucleotide which hybridizes to the ⁇ bosome binding region or the coding region of the Brn-3b mRNA
  • oligonucleotide is delivered which hybridizes to the Brn-3b mRNA
  • Antisense ohgonucleotides are postulated to inhibit target gene expression by interfering with one or more aspects of RNA metabolism including processing, translation and metabolic turnover
  • Chemically modified ohgonucleotides may be used and may enhance resistance to nucleases and/or cell permeability Antisense
  • An inhibitor of Brn-3b comprises a polynucleotide which can hybridize to the Brn-3b mRNA Typically such a polynucleotide will be an RNA molecule Such a polynucleotide may hyb ⁇ dize to all or part of the Bra-3b mRNA Typically the polynucleotide will be complementary to all of or a region of the Brn-3b mRNA For example, the polynucleotide may be the exact complement of all or a part of Brn-3b mRNA However, absolute complementarity is not required and preferred polvnucleotides which have sufficient complementa ⁇ ty to form a duplex having a melting temperature of greater than 40 °C under physiological conditions are particularly suitable for use in the present invention
  • the polynucleotide may be a polynucleotide which hybridises to the Brn-3b mRNA under conditions of medium to high st
  • the polynucleotide hybridizes to the region of the mRNA corresponding to the coding sequence defined by nucleotides 213 to 392 of SEQ ID NO 1
  • the polynucleotide may hybridize to all or part of this region
  • a polynucleotide may be employed which hybridises to all or part of the 5'- or 3 '-untranslated region of the mRNA These regions correspond to nucleotides 1 to 212 and 1446 to 31 0 of SEQ LD NO 1
  • the polynucleotide will typically be at least 40, for example at least 60 or at least 80, nucleotides in length and up to 100, 200, 300, 400 500, 600 or 700 nucleotides in length or even up to a few nucleotides, such as five or ten nucleotides, shorter than SEQ LD NO 1
  • the polynucleotide is an antisense RNA it may be expressed in a cell from a recombinant
  • Promoters/enhancers and other expression regulation signals may be selected to be compatible with the host cell for which the expression vector is designed
  • mammalian promoters such as b-actin promoters
  • Tissues-specific promoters in particular neuronal cell specific promoters (for example the tyrosme hydroxylase (TH), L7, or neuron specific enolase (NSE) promoters)
  • TH tyrosme hydroxylase
  • NSE neuron specific enolase
  • Viral promoters may also be used, for example the Moloney mu ⁇ ne leukaemia virus long terminal repeat (MMLV LTR), the promoter rous sarcoma virus (RSV) LTR promoter, the SV40 promoter, the human cytomegalovirus (CMV) IE promoter, herpes simplex virus promoters or adenovirus promoters All these promoters are readily available in the art
  • Preferred promoters are tissue specific promoters such as the casein gene promote
  • Vectors may further include sequences flanking the polynucleotide giving ⁇ se to antisense RNA which comp ⁇ se sequences homologous to eukaryotic genomic sequences, preferably mammalian genomic sequences, or viral genomic sequences
  • a plasmid vector comp ⁇ sing the expression cassette flanked by viral sequences for example HSV1 or HSV2 sequences
  • a viral vector fpr example an HSV vector, suitable for dehve ⁇ ng the polynucleotides of the invention to a mammalian cell
  • suitable viral vectors include retroviruses, including lentiviruses, adenoviruses and adeno-associated viruses Gene transfer techniques using these viruses are will known to those skilled in the art Retrovirus vectors for example may be used to stably integrate the polynucleo
  • Antisense ohgonucleotides An antisense oligonucleotide will typically have a sequence such that it will bind to the Brn-3b mRNA Therefore it will typically have a sequence which is the complement of a region of the sequence shown in SEQ LD NO 1
  • An antisense oligonucleotide will generally be from 6 to 40 nucleotides in length Preferably it will be from 12 to 20 nucleotides in length Generally the oligonucleotide used will have a sequence that is absolutely complementary to the target sequence
  • absolute complementa ⁇ ty may not be required and in general any oligonucleotide having sufficient complementa ⁇ ty to form a stable duplex (or triple helix as the case may be) with the target nucleic acid is considered to be suitable
  • the stability of a duplex (or triplex) will depend / «ter aha on the sequence and length of the hybridizing oligonucleotide and the degree of complementa ⁇ ty between
  • Antisense ohgonucleotides may be chemically modified
  • phosphorothioate ohgonucleotides may be used
  • Other deoxynucleotide analogs include methylphosphonates, phosphoramidates, phosphorodithioates, N3'P5'-phosphoram ⁇ dates and oligo ⁇ bonucleotide phosphorothioates and their 2'-0-alkyl analogs and 2'-0-methyl ⁇ bonucleot ⁇ de methylphosphonates
  • MBOs Mixed backbone ohgonucleotides
  • MBOs contain segments of phosphothioate o godeoxynucleotides and approp ⁇ ately placed segments of modified o godeoxy- or oligo ⁇ bonucleotides
  • MBOs have segments of phosphorothioate linkages and other segments of other modified ohgonucleotides, such as methylphosphonate, which is non-ionic, and very resistant to nucleases or 2'-0-alkylohgo ⁇ bonucleot ⁇ des
  • the vectors and antisense ohgonucleotides of the invention optionally with an additional therapeutic polypeptide or nucleic acid/vector encoding said therapeutic polypeptide, may thus be administered to a human or animal in need of treatment Cancers which may be treated using the vectors, viral strains, antisense ohgonucleotides and compositions of the invention mclude breast or ovarian cancer and, in particular, breast or
  • the antisense ohgonucleotides and compositions comp ⁇ sing antisense ohgonucleotides of the invention together may be administered by direct miection into the site to be treated, for example mammary tissue
  • the antisense ohgonucleotides are combined with a pharmaceutically acceptable carrier or diluent to produce a pharmaceutical composition
  • Suitable earners and diluents include isotonic saline solutions, for example phosphate-buffered saline
  • the composition may be formulated for parenteral, intramuscular, intravenous, subcutaneous, intraocular or transdermal administration
  • the dose at which an antisense oligonucleotide is administered to a patient will depend upon a variety of factors such as the age weight and general condition of the patient, the cancer that is being treated and the stage which the cancer has reached, and the particular antisense oligonucleotide that is being administered
  • a suitable dose may however be from 0 1 to 100 mg/kg body weight such as 1 to 40 mg/kg body weight
  • polynucleotides giving ⁇ se to antisense RNA of the invention may be administered directly as a naked nucleic acid construct
  • Uptake of naked nucleic acid constructs by mammalian cells is enhanced by several known transfection techniques for example those including the use of transfection agents
  • transfection agents include catiomc agents (for example calcium phosphate and DEAE-dextran) and pofectants (for example pofectam and transfectam )
  • nucleic acid constructs are mixed with the transfection agent to produce a composition
  • the naked nucleic acid construct, viral vector comprising the polynucleotide or composition is combined with a pharmaceutically acceptable earner or diluent to produce a pharmaceutical composition
  • Suitable carriers and diluents include isotonic saline solutions, for example phosphate-buffered saline
  • the composition may be formulated for parenteral, intramuscular, intravenous, subcutaneous, intraocular or transdermal administration
  • the pharmaceutical composition is administered in such a way that the polynucleotide of the invention, viral vector for gene therapy, can be incorporated into cells at an approp ⁇ ate area
  • the amount of virus administered is m the range of from 10 to 10 pfu, preferably from 10 to 10 7 pfu, more preferably about 10 6 pfu for herpes viral vectors and from 10 to 10 pfu, preferably from 10 7 to 10 pfu, more preferably about 10 pfu for adeno viral vectors.
  • typically 1-2 ml of virus in a pharmaceutically acceptable suitable carrier or diluent is administered.
  • the amount of nucleic acid administered is typically in the range of from 1 ⁇ g to 10 mg.
  • Such a system may, for example, involve administering the antibiotic tetracycline, to activate gene expression via its effect on the tet repressor/NP16 fusion protein.
  • tissue-specific promoters will be of assistance in the treatment of disease using the polypeptides, polynucleotide and vectors of the invention.
  • tissue-specific promoters will be of assistance in the treatment of disease using the polypeptides, polynucleotide and vectors of the invention.
  • tissue-specific promoters will be of assistance in the treatment of disease using the polypeptides, polynucleotide and vectors of the invention.
  • tissue-specific promoters will be of assistance in the treatment of disease using the polypeptides, polynucleotide and vectors of the invention.
  • tissue-specific promoters will be of assistance in the treatment of disease using the polypeptides, polynucleotide and vectors of the invention.
  • several neurological disorders are due to aberrant expression of particular gene products in only a small subset of cells. It will be advantageous to be able express therapeutic genes in only the relevant affected cell types, especially where such genes are toxic when expressed in other cell types.
  • the invention provides a method for identifying an inhibitor of Brn-3b expression comprising: (i) providing a test construct comprising a Brn-3b promoter operably linked to a coding sequence; (ii) contacting a substance to be tested with the test construct under conditions that would permit expression of the polypeptide encoded by the said coding sequence to be expressed in the absence of the said substance; and (iii) determining whether the said substance inhibits the expression of Brn-3b.
  • any suitable assay format may be used for identifying an inhibitor of Brn-3b expression.
  • the assay is carried out using a cell harbouring a promoter reporter polypeptide construct.
  • a typical assay is as follows: - a defined number of cells are inoculated, in for example lOO ⁇ l of growth medium, into the wells of a plastics micro-titre plate in the presence of a substance to be tested
  • OD optical density
  • any reporter polypeptide may be used, for example GUS or GFP are used GUS is assayed by measu ⁇ ng the hydrolysis of a suitable substrate, for example 5-bromo-4-chloro-3- indolyl- ⁇ -D-glucoromc acid (X-gluc)or 4-methylumbelhferyl- ⁇ -glucuronide (MUG)
  • X-gluc 5-bromo-4-chloro-3- indolyl- ⁇ -D-glucoromc acid
  • MUG 4-methylumbelhferyl- ⁇ -glucuronide
  • the coding sequence may be the Brn-3b coding sequence itself
  • a mammary cancer cell line which exhibits Brn-3b overexpression could be used
  • the expression of Brn-3b may be followed by for example, Northern/RNA blotting, Western/antibody blotting, RNA in situ hybridization or lmmunolocalisation
  • the invention further provides a method for identifying an inhibitor of Brn-3b activity comprising
  • Suitable Brn-3b for the assay can be obtained, for example, recombinantly by any method known to those skilled in the art Any suitable format may be used for identifying an inhibitor ofBrn-3b
  • the substance may be tested with any other known transcnption factor to exclude the possibility that the test substance is a general inhibitor of transc ⁇ ption factors activity
  • the reaction mixture can contain a suitable buffer
  • a suitable buffer includes anv suitable biological buffer that can provide buffe ⁇ ng capability at a pH conducive to the reaction requirements of the Brn-3b polypeptide
  • a substance which inhibits the expression of Brn-3b may do so by binding directly to the promoter, thus preventing the initiation of transcription
  • a substance could bind to a protein which is associated with the promoter and is required for transc ⁇ ption This may result in reduced levels of transcription
  • the Brn-3b promoter reporter polypeptide constructs of the invention may include the untranslated region of the Brn-3b gene Therefore a substance may reduce Brn-3b expression by binding to the untranslated region of the Brn-3b gene This could prevent the initiation of translation Alternatively a substance could bind to a protein associated with the untranslated region and prevent the protein associating with the untranslated region
  • a substance which inhibits the activity of Brn-3b may do so by binding to one or both of the enzymes
  • Such enzyme inhibition may be reversible or irreversible
  • An l ⁇ eversible inhibitor dissociates very slowly from its target enzyme because it becomes very tightly bound to the enzyme, either covalently or non-covalently Reversible inhibition, in contrast with meversible inhibition, is characterised by a rapid dissociation of the enzyme-mhibitor complex
  • the test substance may be a competitive inhibitor
  • the enzyme can bind substrate (forming an enzyme-substrate complex) or inhibitor (enzyme-inhibitor complex) but not both Many competitive inhibitors resemble the substrate and bind the active site of the enzyme The substrate is therefore prevented from binding to the same active site
  • a competitive inhibitor diminishes the rate of catalysis by reducing the proportion of enzyme molecules bound to a substrate
  • the inhibitor may also be a non-competitive inhibitor
  • non-competitive inhibition which is also reversible, the inhibitor and substrate can bind simultaneously to an enzyme molecule This means that their binding sites do not overlap
  • a non-competitive inhibitor acts by decreasing the turnover number of an enzyme rather than by diminishing the proportion of enzyme molecules that are bound to substrate
  • the inhibitor can also be a mixed inhibitor Mixed inhibition occurs when an inhibitor both effects the binding of substrate and alters the turnover number of the enzyme
  • a substance which inhibits the activity of Brn-3b may also do so by binding to the substrate
  • the substance may itself catalyze a reaction of the substrate, so that the substrate is not available to the enzyme.
  • the inhibitor may simply prevent the substrate binding to the enzyme.
  • Suitable candidate substances include antibody products (for example, monoclonal and polyclonal antibodies, single chain antibodies, chimaeric antibodies and CDR-grafted antibodies) which are specific for Brn-3b
  • antibody products for example, monoclonal and polyclonal antibodies, single chain antibodies, chimaeric antibodies and CDR-grafted antibodies
  • combinatorial libraries, defined chemical entities, peptide and peptide mimetics, ohgonucleotides and natural product libraries may be screened for activity as inhibitors of Brn-3b in assays such as those described below.
  • the candidate substances may be chemical compounds.
  • the candidate substances may be used in an initial screen of, for example, ten substances per reaction, and the substance of these batches which show inhibition tested individually.
  • An inhibitor of Brn-3b expression and/or activity is one which produces a measurable reduction in Brn-3b expression and/or activity in the assays described above.
  • Preferred substances are those which inhibit Brn-3b expression and/or activity by at least 10%, at least 20%, at least 30%, at least 40% at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99% at a concentration of the inhibitor of 1 ⁇ g ml " ', lO ⁇ g ml "1 , lOO ⁇ g ml " ', 500 ⁇ g ml " ', lmg ml "1, lOmg ml " ', lOOmg ml " '.
  • the percentage inhibition represents the percentage decrease in expression/activity in a comparison of assays in the presence and absence of the test substance. Any combination of the above mentioned degrees of percentage inhibition and concentration of inhibitor may be used to define an inhibitor of the invention, with greater inhibition at lower concentrations being prefe ⁇ ed.
  • Candidate substances which show activity in assays such as those described below can then be tested on mammary cancer cell lines for example.
  • Candidate inhibitors could be tested for their ability to attenuate Bm-3b expression in mammary cancer cell lines in which Brn-3b is up-regulated and also for the effect on BRCA-1 expression in mammary cancer cell lines in which BRCA-1 is down-regulated.
  • Inhibitors of Brn-3b expression and/or activity identified by the screening procedures described above may be used to treat breast or ovarian cancer and, in particular, breast or ovarian cancer in which Brn-3b expression is up-regulated such as non-familial breast cancer.
  • the condition of a patient suffering from a cancer can therefore be improved by administration of such an inhibitor.
  • a therapeutically effective amount of such an inhibitor may be given to a human patient in need thereof
  • an inhibitor for use in preventing or treating breast or ova ⁇ an cancer will depend upon factors such as the nature of the substance identified, whether a pharmaceutical or veterinary use is intended, etc Typically an inhibitor is formulated for use with a pharmaceutically acceptable carrier or diluent For example it may be formulated for topical, parenteral, intravenous, intramuscular, subcutaneous, intraocular, transdermal or oral administration A physician will be able to determine the required route of administration for each particular patient The pharmaceutical earner or diluent may be, for example, an isotonic solution The dose of inhibitor may be determined according to va ⁇ ous parameters, especially according to the substance used, the age, weight and condition of the patient to be treated, the route of administration, and the required regimen Again, a physician will be able to determine the required route of administration and dosage for any particular patient The following Example illustrates the invention EXAMPLE
  • RNA from normal mammary gland and from malignant breast tumours was obtained from the Candis Tissue Bank Research Centre Liverpool University Reverse transcriptase/polymerase chain reaction assay
  • RNA from each sample was used as a template for cDNA synthesis
  • Th synthesized cDNA was used in RT-PCR assays as previously desc ⁇ bed (17) using the following oligonucleotide pnmers Brn-3a 5' GTCGACATGGACTCGGACACG-3', 3'- ACGGTGAATGACTCCCCCGA-5 1 , Brn-3b 5'GGAGAAGAAGCGCAAGC-3', 3'CTGAGAACCGGAGAGGTCT-5'
  • Amplification of the lnva ⁇ antly expressed human cyclophihn mRNA used as a control was earned out in parallel using the following primers 5'- TTGGGCCGCGGTACTCCTTTCA-3 1 , and 3'-TTTCGTATGGCCCAGGACCG-5'
  • 20 ⁇ l of each PCR product was fractionated on a 2% agarose gel and blotted onto Hybond-N+ nylon membrane (Amersham International, Little Chalfont, United Kingdom
  • the Bm-3a and Brn-3b expression vectors contain full length cDNA clones under the control of the moloney murme leukaemia virus promoter and have previously been described (7 17)
  • the BRCA-1 promoter/reporter constructs contain 4 kilo-bases or 400 bases of upstream sequence containing the BRCA-1 ⁇ and ⁇ promoters cloned into the pGL2 luciferase vector Transient transfection
  • MCF7 cells were routinely grown in Dulbecco's modified Eagle's medium containing L- glutamate and phenol red which was supplemented with 10% foetal calf serum and lOng of insulin per ml Before expenments were earned out subconfluent cells were maintained in phenol red-free Dulbecco's modified Eagle's medium containing 10% dextran coated charcoal- st ⁇ ped foetal calf serum prepared according to the method desc ⁇ bed by Mighaccio et al , (18) and lOng of insulin per ml for 72 hours The medium was replaced by 5 ml of fresh medium 12 hours prior to transfection Transfection of plasmid DNA was earned out according to the method of Gorman (19) Routinely 5 ⁇ g of reporter DNA and 5 ⁇ g of each expression vector were transfected into 5xl0 3 cells and the cells harvested after 72 hours The amount of DNA taken up by the cells in each case was measured by slot blotting of 15 ⁇ l of the extract and hybridization with a probe
  • HPV activating transcription factor Brn-3 a is over expressed in CLN3 lesions Journal of Clinical Investigation 101 1687-1692, 1998
  • the POU domain factors Brn-3 a and Brn-3b interact with the estrogen receptor and differentially regulate transc ⁇ ptional activity via an ERE Molecular and Cellular Biology 18 1029-1041, 1997

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Abstract

La présente invention concerne un inhibiteur de l'expression et/ou de l'activité du Brn-3b, lequel inhibiteur convient particulièrement au traitement du cancer de sein ou du cancer des ovaires. L'invention concerne également un procédé aboutissant à l'identification d'un inhibiteur du Brn-3b. Ce procédé consiste (a) à prendre une construction test comprenant un promoteur du Brn-3b fonctionnellement lié à une séquence codante. Le procédé consiste ensuite (b) à mettre en contact avec la construction test une substance à tester dans les conditions qui permettraient au polypeptide codé par la séquence codante considérée de s'exprimer en l'absence de la substance considérée. Le procédé consiste enfin (c) à déterminer si la substance considérée inhibe l'expression du Brn-3b.
EP99946334A 1998-09-14 1999-09-14 Inhibiteur du brn-3b et son utilisation dans le traitement contre le cancer du sein et le cancer des ovaires Withdrawn EP1112356A1 (fr)

Applications Claiming Priority (3)

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GB9819999 1998-09-14
GBGB9819999.5A GB9819999D0 (en) 1998-09-14 1998-09-14 Treatment of cancer
PCT/GB1999/003047 WO2000015780A1 (fr) 1998-09-14 1999-09-14 Inhibiteur du brn-3b et son utilisation dans le traitement contre le cancer du sein et le cancer des ovaires

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EP1112356A1 true EP1112356A1 (fr) 2001-07-04

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EP (1) EP1112356A1 (fr)
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AU (1) AU5874899A (fr)
CA (1) CA2343354A1 (fr)
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WO (1) WO2000015780A1 (fr)

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