WO2000034466A1 - Traitement du cancer du col de l'uterus - Google Patents

Traitement du cancer du col de l'uterus Download PDF

Info

Publication number
WO2000034466A1
WO2000034466A1 PCT/GB1999/004116 GB9904116W WO0034466A1 WO 2000034466 A1 WO2000034466 A1 WO 2000034466A1 GB 9904116 W GB9904116 W GB 9904116W WO 0034466 A1 WO0034466 A1 WO 0034466A1
Authority
WO
WIPO (PCT)
Prior art keywords
brn
product
expression
hpv
candidate substance
Prior art date
Application number
PCT/GB1999/004116
Other languages
English (en)
Inventor
David Seymour Latchman
Vishwanie Budhram-Mahadeo
Daniel Ndisang
Original Assignee
University College London
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Priority claimed from GBGB9826888.1A external-priority patent/GB9826888D0/en
Application filed by University College London filed Critical University College London
Priority to EP99959533A priority Critical patent/EP1151094A1/fr
Publication of WO2000034466A1 publication Critical patent/WO2000034466A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—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
    • C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating activity
    • 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
    • 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
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/708—Specific hybridization probes for papilloma
    • 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
    • C12Q2600/136—Screening for pharmacological compounds

Definitions

  • the invention relates to the diagnosis or treatment of cancer and to screening for individuals at risk from cancer.
  • viruses to infect successfully an individual or to have other detrimental effects, such as causing oncogenesis is determined by many factors. Such factors include the dose of virus which the host receives, the resistance of the host, the route of infection and the virulence of the virus .
  • HPV- 16 and HPV-18 Human papillomavirus types 16 and 18 (HPV- 16 and HPV-18) are thought to play a part in causing cervical cancer in infected individuals. However HPV- 16 and HPVrl ⁇ are found both in women with cervical cancer and in women with undetectable or minimal cervical abnormality. A polymorphism in a human protein has been shown to affect susceptibility to cervical cancer caused by HPV. Individuals with a particular polymorphism in the p53 tumour suppressor protein are found to have an increased risk of cervical cancer caused by HPV.
  • Brn-3a is over 300 fold higher in cervical intraepithelial neoplasia (CIN)lesions than in normal cervical material.
  • Brn-3a is a member of the POU family of transcription factors and is expressed in neuronal cells and in cervical cells.
  • non-malignant cervical cells in the individuals with CLN lesions also had high levels of expression of Brn-3a. This latter finding suggested that the levels of Brn-3a may not be important in determining whether a cervical cell became malignant or not. This work has now been published (1).
  • an antisense-based approach can be used to reduce levels of Brn-3a in a transformed cervical cell line, and that this causes a reversal of the malignant phenotype.
  • the cells exhibit a reduced cellular growth rate, a reduced saturation density and a reduced ability to grow in an anchorage independent manner.
  • the invention therefore provides a product that binds, causes a decrease in intracellular levels of, or inhibits the activity of Brn-3a for use in the treatment, prevention or diagnosis of a cervical cancer attributable to HPV.
  • the invention further provides method of identifying a product that binds to Brn-3a comprising contacting Brn-3a, or a mimic of Brn-3a that can bind to a Brn-3a specific antibody, with a candidate substance and determining whether the candidate substance binds Brn-3a or the mimic of Brn-3a.
  • the invention also provides a method of identifying a product that causes a decrease in intracellular levels of Brn-3a comprising contacting a candidate substance with a cell or cell extract and determining whether the candidate causes a decrease in intracellular levels.
  • the invention further provides a method of identifying a product that inhibits the activity of Bra-3a comprising (i) contacting Brn-3a, or a mimic of Brn-3a that can bind to a Brn-3a specific antibody, with a candidate substance and determining whether the candidate substance binds Brn-3a or the mimic of Brn-3a; or (ii) contacting an agent that can bind to Brn-3a with a candidate substance and determining whether the candidate substance binds to the agent; or (iii)(a) contacting Brn-3a, or a mimic of Brn-3a with Brn-3a activity, with a candidate substance under conditions that would permit activity of Brn-3a, and (b) determining whether the candidate substance inhibits the activity of Brn-3a or the mimic ofB
  • the product may be used to treat cancer and therefore the invention provides a method of treating a host suffering from cancer comprising administering to the host a therapeutically effective amount of the product.
  • Figure 1 shows results of reverse transcriptase/ polymerase chain amplification of either the Brn-3a (A) Brn-3b (B) or control cyclophilin (C) mRNAs in three samples obtained from women with no histoiogicaly detectable cervical abnormality (N) or three samples from CLN3 lesions (3).
  • the positive control of cDNA prepared from human neuroblastoma mRNA (+) and the negative control of no cDNA (-) are also indicated.
  • Figure 2 shows Brn-3b mRNA levels in cervical samples from women with no histoiogicaly detectable abnormality (N), from CIN3 regions (CLN3) or from normal samples adjacent to a CIN3 lesion (N-CIN).
  • N histoiogicaly detectable abnormality
  • CLN3 CIN3 regions
  • N-CIN normal samples adjacent to a CIN3 lesion
  • the horizontal bar shows the mean in each group and the number of samples in each group is indicated (n).
  • Figure 3 shows Brn-3a mRNA levels in the same samples as in figure 2.
  • Figure 4 shows Brn-3b protein levels as determined by Western blotting of cervical samples from women with no histoiogicaly detectable abnormality (N), from CLN3 samples (CLN3) or from normal samples adjacent to a CLN3 lesion (N-CIN).
  • Figure 5 Brn-3a protein levels in the same samples as in Figure 4.
  • Figure 6 shows levels of Brn-3a protein in parental SiHa (panel a) or C-33 cells (panel b) cells or clonal cell lines transfected with expression vector lacking any insert, (Pci-neo) Brn-3a expression vector, (Brn-3a) Brn-3b expression vector (Brn- 3b) or a vector expressing the antisense strand of the Brn-3a gene (-3 A) and then grown in the presence or absence of dexamethasone (dex) Each data point shows a different independently isolated, clonal cell line.
  • Figure 7 shows levels of HPV protein in parental SiHa cells or clonal cell lines transfected with expression vector lacking any insert, (neo) Brn-3a expression vector (A), Brn-3b expression vector (B), or two different cell lines transfected with the Brn-3a anti sense vector (-3 A clone 3 and -3 A clone 5).
  • Figure 8 shows cell growth curves of SiHa cells. The growth rate of parental
  • SiHa cells is compared to that of SiHa cells transfected with empty expression vector (neo) Brn-3a expression vector, Brn-3b expression or the antisense Bm-3a vector (- 3 A) in the presence or absence or dexamethasone.
  • Figure 9 shows cell growth curves of C-33 cells.
  • the growth rate of parental C-33 cells is compared to that of C-33 cells transfected with empty expression vector (neo), Brn-3a expression vector, Brn-3b expression or the antisense Brn-3a vector in the presence or absence of dexamethasone.
  • Figure 10 shoes saturation density of parental SiHa cells or SiHa - derived clones stably transfected with empty expression vector (neo), Brn-3a expression vector, Brn-3b expression vector or two cell lines(-3A clone 3 and -3 A clone 5) transfected with the antisense Brn-3a vector and grown in the presence or absence of dexamethasone.
  • Figure 11 shows saturation density of parental C-33 cells or C-33 -derived clones stably transfected with empty expression vector (neo), Bm-3a expression vector, Brn-3b expression vector or the antisense Bm-3a vector (-3 A) and grown in the presence or absence of dexamethasone.
  • Figure 12 shows anchorage independent growth as assayed by colony forming efficiency CFE (number of colonies formed/ number of colonies seeded XI 00). The result is show for parental SiHa cells and SiHa-derived clonal cell lines transfected with empty expression vector, Brn-3a expression vector, Brn-3b expression vector or the Brn-3b antisense vector (panel a) or for parental C-33 cells and similarly transfected stable cell lines derived from C-33 (panel b). Values are the mean of three separate determinations.
  • Figure 13 shows the volume of the tumour formed at the indicated day after injection of nude mice with SiHa cells stably transfected with empty expression vector (group 1, Gpl, open squares) or with SiHA cells stably transfected with the Brn-3a anti-sense construct (group 2, Gp2, solid diamonds). Values are the means of six injected animals whose standard deviation is shown by the bars.
  • the cancer which is prevented, treated, diagnosed, or susceptibility to which can be screened for, in the invention is a cervical cancer attributable to HPV.
  • the cancer is one in which the malignant state of the cell is maintained by the expression of HPV proteins in the cell such as the HPV proteins E6 or E7.
  • the expression of the HPV proteins is generally dependent on the presence of Brn-3a in the cell. Therefore, typically the cancer is one in which malignant cells comprise HPV virus or HPV genome.
  • the HPV genome is generally integrated into the genome of the cell.
  • the HPV is typically HPV-16 or HPV-18.
  • the levels of Brn-3a are elevated in the malignant cell.
  • a cervical cell which has elevated Brn-3a levels is one which has a higher level of Brn-3a than the average (mean) level of Brn-3a in the non-malignant cervical cells of females in a population sample.
  • the Brn-3a or Brn-3a mRNA levels are at least 10-, 20-, 50-, 100-, 300- or 500-fold those of the average levels.
  • the ratio of levels of Brn-3a mRNA to cyclophilin mRNA is greater than from 0.1 : 1, for example greater than 0.2, 0.4, 0.6 or 0.8: 1.
  • any suitable product which binds Brn-3a may be used in the invention.
  • the product binds Brn-3a specifically.
  • Such a product may or may not inhibit the activity of Brn-3a.
  • the product may bind reversibly or irreversibly to Brn-3a.
  • Reversible binding in contrast with irreversible binding, is characterised by a rapid dissociation of the Brn-3 a/product complex.
  • the product may resemble a natural agent that binds Brn-3 a either in its structure or binding characteristics.
  • Such an agent is typically a cellular or HPV component that binds Brn-3a.
  • the agent is a polynucleotide or a polypeptide the product may have homology with the natural agent.
  • the product may bind Brn-3a at the same site as the agent binds.
  • Such a product is typically able to compete for, or inhibit, the binding of the agent to Brn-3 a.
  • the product does not bind Brn-3 a at a site that overlaps with the site at which the agent binds.
  • such a product does not compete with the agent for binding to Brn-3a, but may or may not inhibit the binding of the agent to Brn-3a
  • the product may or may not be able to bind Bm-3a at the same time as the agent binds Brn-3a
  • the product only binds Brn-3a when Brn-3 a is bound to the agent
  • Any suitable product that inhibits the activity of Brn-3a may be used in the present invention
  • Such a product may bind Brn-3a, typically with binding characteristics as discussed above
  • the product inhibits the activity of Brn- 3a in a specific or substantially specific manner
  • the product may or may not cause a change in the structure of Brn-3 a
  • the inhibitor of Brn-3a activity causes Brn-3a to change to a less active or non-functional form
  • the change may be reversible or irreversible
  • Brn-3a only adopts such a changed form when bound to the product
  • An irreversible change may occur, for example, if Brn-3 a is chemically modified or is broken down by the product, for example by the breaking of peptide bonds
  • a product which binds and inhibits the activity of Brn-3 a typically does so by
  • the product inhibits the activity of Brn-3 a in a cell not by binding Brn-3a, but by binding a cellular or HPV component
  • the product may bind and/or act on the component in the same manner as the product described above binds and acts upon Brn-3a
  • the binding of the product to the component is typically specific and may be reversible or irreversible, and may or may not cause a change in the structure of the component
  • Such a product typically resembles the structure and/or binding characteristics of Brn-3 a, and therefore the product may be act as a 'mimic' of Brn-3 a which has a lower activity than Brn-3 a
  • the product may bind the component at the same site, at an cellular or HPV component which naturally binds Brn3a, and/or (n) changing the structure of Brn-3a so that it has a decreased ability to 'activate' or direct a Brn-3 a dependent effect mediated by the component
  • the product inhibits the activity of Brn-3
  • the product may or may not compete with Brn-3 a to bind the component
  • the product may or may not inhibit the binding of the component with Brn-3 a
  • the product binds the component without causing activation of the component or causes less activation than Brn-3a.
  • the product which binds the component may be a peptide which has homology with Brn-3a but which has no Brn-3a activity or a reduced Brn-3a activity.
  • the product has from 10%, 1%, 0.1% or less of the activity of Brn-3a.
  • the cellular component may be a co-activator protein which binds to Brn-3 a, and typically increases the activity of Brn-3 a.
  • the HPV component is one which upon binding Brn-3 a causes an increase in the expression of a factor which contributes or leads to the malignant phenotype of the cell.
  • the HPV component is the upstream regulatory region (URR) of the HPV genome.
  • the component may comprise the ATGCAATT motif of the URR. This motif may play a role in the expression of the oncogenic
  • HPV proteins E6 and E7 HPV proteins E6 and E7.
  • Any suitable product that decreases intracellular levels of Brn-3a may be used in the present invention.
  • Such a product typically inhibits the expression of Brn-3a or increases the breakdown of Brn-3 a.
  • a product which inhibits the expression of Brn-3 a generally inhibits one or more cellular or HPV components that promote the expression of Brn-3 a, or stimulates one or more cellular or HPV components that inhibit the expression of Brn-3a.
  • these components are specific or substantially specific to the expression of Brn-3a.
  • the product may bind and/or act on the component in the same manner as the product described above binds and acts upon Brn-3a.
  • binding of the product to the component is typically specific and may be reversible or irreversible, and may or may not cause a change in the structure of the component.
  • the component may directly or indirectly promote or inhibit the expression of Brn-
  • Cellular components that directly promote expression include the promoter of
  • Brn-3a transcription factors that bind or affect expression from the Brn-3a promoter
  • an RNA poly erase that can express mRNA from the Bm-3a gene
  • nuclear factors that bind to Brn-3a mRNA and/or transport Brn-3a mRNA from the nucleus to the cytoplasm nuclear factors that bind to Brn-3a mRNA and/or transport Brn-3a mRNA from the nucleus to the cytoplasm
  • translation factors that contribute to translating the Brn-3 a mRNA to Brn- 3a protein, or factors that bind and/or transport Brn-3a protein into the nucleus.
  • Components that indirectly promote expression include components that are one step removed from the Brn-3a expression pathway, such as the promoters, transcription factors, polymerases, nuclear factors, translation factors of components that directly promote the expression of Brn-3a. Components that indirectly promote expression may thus be one, two or more steps removed from the components that directly promote expression of Brn-3 a.
  • the product may inhibit transcription or translation of Brn-3a.
  • the product is a specific inhibitor of transcription from the Brn-3a gene, and does not inhibit transcription from other genes.
  • the product may bind to the Br - 3a gene either (i) 5' to the coding sequence, and/or (ii) to the coding sequence, and/or (iii) 3' to the coding sequence.
  • the product may bind to the Brn-3a promoter, and inhibit the initiation of transcription.
  • the product may bind and inhibit the action of a protein which is required for transcription from the Brn-3 a gene.
  • the product may bind to the untranslated or translated regions of the Brn-3a mRNA. This could prevent the initiation of translation. Alternatively the inhibitor could bind to a protein which associates with the untranslated region and prevent the protein associating with the untranslated region.
  • Products which are polynucleotides, such as the antisense polynucleotides discussed below, may be chemically modified. This may enhance their resistance to nucleases and may enhance their ability to enter cells. For example, phosphorothioate oligonucleotides may be used.
  • deoxynucleotide analogs include methylphosphonates, phosphoramidates, phosphorodithioates, N3'P5'- phosphoramidates and oligoribonucleotide phosphorothioates and their 2'-O-alkyl analogs and 2'-O-methylribonucleotide methylphosphonates.
  • MBOs mixed backbone oligonucleotides
  • MBOs contain segments of phosphothioate oligodeoxynucleotides and appropriately placed segments of modified oligodeoxy- or oligoribonucleotides.
  • MBOs have segments of phosphorothioate linkages and other segments of other modified oligonucleotides, such as methylphosphonate, which is non-ionic, and very resistant to nucleases or 2'- O-alkyloligoribonucleotides.
  • the expression of Brn-3a in a cell may be reduced by the presence in that cell of a product which can bind to the Brn-3a mRNA. Therefore a polynucleotide which is capable of hybridizing to Brn-3 a mRNA can constitute an appropriate inhibitor of Brn-3 a expression.
  • the polynucleotide may be antisense to the Brn-3a mRNA. Such a polynucleotide may be capable of hybridising to Brn-3 a mRNA and may thus inhibit the expression of Brn-3a by interfering with one or more aspects of Brn-3a mRNA metabolism including transcription, mRNA processing, mRNA transport from the nucleus, translation or mRNA degradation.
  • the antisense polynucleotide may be DNA, but is typically RNA.
  • the antisense polynucleotide may be provided as single or double stranded polynucleotide.
  • the antisense polynucleotide typically hybridises to the Brn-3 a mRNA to form a duplex (typically an RNA-RNA duplex) which can cause direct inhibition of translation and/or destabilisation of the mRNA.
  • a duplex typically an RNA-RNA duplex
  • Such a duplex may be susceptible to degradation by nucleases.
  • the antisense polynucleotide may hybridize to all or part of the Brn-3a mRNA. Typically the antisense polynucleotide hybridizes to the ribosome binding region or the coding region of the Brn-3 a mRNA.
  • the polynucleotide may be complementary to all of or a region of the Brn-3a mRNA.
  • the polynucleotide may be the exact complement of all or a part of Brn-3a mRNA.
  • absolute complementarity is not required and polynucleotides which have sufficient complementarity to form a duplex having a melting temperature of greater than 20°C, 30°C or 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-3 a mRNA under conditions of medium to high stringency such as 0.03M sodium chloride and 0.03M sodium citrate at from about 50 to about 60 degrees centigrade. It is preferred that the polynucleotide hybridizes to all or part of the region of the Brn-3 a mRNA corresponding to the coding sequence defined by nucleotides 1 to 1272 of SEQ ID NOT.
  • the antisense polynucleotide sequence is complementary to the entire coding sequence of the mRNA and to the 50 nucleotides of the mRNA immediately 5' of the coding sequence.
  • the polynucleotide may hybridise to all or part of the 5'- or 3 '-untranslated region of the mRNA.
  • the polynucleotide will typically be from 6 to 40 nucleotides in length. Preferably it will be from 12 to 20 nucleotides in length.
  • the polynucleotides may be at least 40, for example at least 60 or at least 80, nucleotides in length and up to 100, 200, 300, 400, 500, 1000, 2000 or 3000 or more nucleotides in length.
  • the length of the antisense oligonucleotide is the same as SEQ ID NO: 1 or up to a few nucleotides, such as five or ten nucleotides, shorter than SEQ ID NO:
  • the invention may be carried out by administering a substance which provides a product with any of the above properties in vivo.
  • a substance is also included in the term 'product'.
  • the substance provides the product extracellularly or intracellularly, such as in the cytoplasm or nucleus.
  • the substance provides the product only in a malignant cell or in a cell with the characteristics of a malignant cell, such as a cell with a high rate of cell division.
  • the substance may provide the product in a tissue specific manner, such as only in cervical cells.
  • the substance may provide the product in the region of a malignant or cervical cell.
  • the substance is an inactive or precursor form of the product which can be processed in vivo to provide the product.
  • the substance may comprise the product associated, covalently or non-covalently, with a carrier.
  • the substance can typically be modified or broken down to provide the product.
  • the substance may, for example, be a polynucleotide which is processed, for example transcribed and/or translated to provide a product as discussed above.
  • a product which binds Brn-3a or Brn-3a mRNA can be used to treat, prevent or diagnose cancer or to determine whether an individual is susceptible to cancer.
  • the finding of elevated levels of Brn-3 a or Brn-3 a mRNA in a cell suggest that the cell is a malignant cell or is at risk of becoming a malignant cell. Measuring the levels of Brn-3a or Brn-3a mRNA in a cell can therefore be used to diagnose cancer or to determine susceptibility to cancer.
  • the levels of Brn-3a or Brn-3a mRNA may be measured in vivo or in vitro.
  • the invention provides a means for measuring the level of Brn-3a or for typing individuals who are predisposed to expressing elevated levels of Brn-3a or Brn-3a mRNA for use in a method of diagnosis of cervical cancer or determining susceptibility to cervical cancer.
  • the invention also provides a method of diagnosing cervical cancer in a female or identifying a female who is susceptible to cervical cancer comprising determining whether cervical cells taken from the female express, or are predisposed to expressing, elevated levels of (i) Brn-3a, or (ii) Brn-3a mRNA.
  • a product which binds to Brn-3a or Brn-3a mRNA can be used to measure levels of Brn-3a or Brn-3a mRNA in a cell. Generally such a product is labelled directly, or can be labelled indirectly.
  • the product is used to measure the levels of Brn-3a or Brn-3a mRNA in cervical cells of a female human being.
  • cervical cells are obtained from the female, typically as a cervical smear.
  • females in a population can be screened to diagnose cervical cancer or determine susceptibility to cervical cancer.
  • HPV virus may also be determined in such screen.
  • the accumulation in cells of a product that binds Brn-3a or Brn-3a mRNA can be used as a marker for cells in which Brn-3a levels are elevated.
  • the product can be used to direct an agent or effect to a cell in which the expression of Bm-3a is elevated.
  • the agent or effect may be therapeutic to the cell, typically contributing to the reversal of a malignant phenotype.
  • Such an agent may bind to the product and typically inhibits the activity of Bm-3a if the product is also bound to Brn-3a
  • the agent or effect may be detrimental to the cell, typically inhibiting the growth or replication of the cell, or killing the cell
  • the agent is npically a toxin
  • the effect is typically electromagnetic radiation
  • a product that inhibits the expression of, causes a decrease in intracellular levels of or inhibits the activity Brn-3a can be used to prevent or treat the cancers discussed above
  • the invention provides a method of identifying a product that causes a decrease in the intracellular levels of Brn-3a comprising providing a candidate substance to a cell or cell extract and determining whether the candidate causes a decrease in intracellular levels
  • the cell may be any of the mammalian cells discussed herein which express Brn-3a
  • the invention provides a method of identifying a product that inhibits expression of Brn-3 a comprising providing a candidate substance to one or more components of the intracellular expression pathway of Bm-3a, or functional analogues of these components, and determining whether (I) the candidate substance binds or inhibits component(s) that promote the expression of Brn-3 a, or (u) the candidate substance stimulates component(s) that inhibit the expression of Brn-3 a
  • component' includes the natural component or a functional analogue of the component
  • the product may be identified by providing a candidate substance to the component and determining whether the candidate substance binds the component Any suitable binding assay format can be used, such as the formats discussed below
  • the product is identified by providing a candidate substance to the component under conditions that permit activity of the component, and determining whether the candidate substance inhibits or stimulates the activity of the component.
  • the component of the cellular expression pathway used in the method is specific or substantially specific to the expression of Brn-3 a.
  • one or more of the following components are used: a Brn-3a promoter, transcription factors that bind or affect expression from the Brn-3a promoter, an RNA polymerase that can express mRNA from the Brn-3 a gene, nuclear factors that bind to Brn-3a mRNA and/or transport Brn-3a mRNA from the nucleus to the cytoplasm, translation factors that contribute to translating the Brn-3 a mRNA to Brn- 3a protein, or factors that bind and/or transport Brn-3a protein into the nucleus.
  • analogues of any of the above components may be used in the method.
  • the analogues will have some or all of the relevant activity of the natural component.
  • the analogues comprise fragments of the natural components.
  • the analogues In the case of components which are polynucleotides or polypeptides the analogues generally have homology with the natural component.
  • the components may be provided from a cell.
  • the components may be inside a cell, typically a recombinant or natural cell in which the components are recombinantly or naturally expressed.
  • the components may be provided in the form of a cell extract or may be purified, or partially purified, from a cell extract.
  • the components are in or from a human cell, for example one which expresses Brn-3a, such as a neuronal cell or cervical cell.
  • the cell may be a mammalian cell, such as a primate or rodent cell, for example a mouse or rat cell.
  • the cell may comprise an HPV genome, typically integrated into the genome of the cell.
  • the cell may be malignant or normal.
  • Cellular or HPV components of the Brn-3 a expression pathway are known or can be readily obtained by the skilled person. They can, for example, be purified from cells based on their ability to bind Brn-3 a or Brn-3 a mRNA.
  • Products which inhibit transcription of Brn-3a can be identified in a method comprising
  • Brn-3a promoter activity operably linked to a second polynucleotide sequence to be expressed in the form of mRNA; (ii) contacting a candidate substance with the test construct under conditions that would permit the second polynucleotide sequence to be expressed in the form of mRNA in the absence of the substance; and (iii) determining whether the substance inhibits expression from the construct.
  • Products which inhibit transcription of Brn-3 a mRNA may be also identified in a method comprising
  • test construct comprising a polynucleotide sequence with Brn-3 a promoter activity operably linked to a coding sequence; (ii) contacting a candidate substance with the test construct under conditions that would permit the polypeptide encoded by the coding sequence to be expressed in the absence of the substance;
  • the polynucleotide with Brn-3 a promoter activity may comprise: (i) the sequence of a human or animal Brn-3a promoter; (ii) a sequence which has homology with (i); or (iii) a sequence which is a fragment of (i) or (ii).
  • the sequence (i) is generally a mammalian Brn-3 a promoter, such as a primate or a rodent, typically a mouse or rat, Brn-3 a promoter.
  • (i) comprises at least from nucleotides -500 to -1, typically -300 to -1 of the Brn-3a gene (the numbers being relative to the transcription start site).
  • the polynucleotide comprises the sequences present in (i) which bind transcription factors or the RNA polymerase, or instead of any of these sequences homologues of the sequences able to bind the same transcription factors and RNA polymerase.
  • sequences or their homologues are present in the polynucleotide in the same order and/or substantially the same relative spacing as in (i).
  • this method is carried out in conditions which in the absence of the test compound lead to expression of the coding sequence from the nucleic acid.
  • the nucleic acid may also comprise other untranscribed or untranslated regions of the Brn-3a gene.
  • the coding sequence typically encodes a protein that is able to act as a reporter of expression.
  • the assay may be carried out in a cell which harbours the nucleic acid.
  • the substance may be tested with any other known promoter to test the possibility that the test substance is a general inhibitor of gene expression.
  • Any reporter polypeptide may be used, for example luciferase, GUS or GFP.
  • Luciferase is assayed by detecting chemiluminescence.
  • GUS is assayed by measuring the hydrolysis of a suitable substrate, for example 5-bromo-4-chloro-3- indolyl- ⁇ -D-glucoronic acid (X-gluc)or 4-methylumbelliferyl- ⁇ -glucuronide (MUG).
  • X-gluc 5-bromo-4-chloro-3- indolyl- ⁇ -D-glucoronic acid
  • MUG 4-methylumbelliferyl- ⁇ -glucuronide
  • the hydrolysis of MUG yields a product which can be measured fluorometrically.
  • GFP is quantified by measuring fluorescence at 590nm after excitation at 494nm.
  • the coding sequence may be the Brn-3 a coding sequence itself, or a fragment of this sequence.
  • the expression of the Brn-3 a may be measured by for example, Northern/RNA blotting, Western/antibody blotting, RNA in situ hybridization or immunolocalisation.
  • Identifying a product that binds to or inhibits the activity of Brn-3a The invention provides a method of identifying products that bind, and which may also inhibit the activity of, Brn-3 a based on the ability of the product to bind
  • Brn-3a Any suitable format may be used for determining whether a product is capable of binding Brn-3a.
  • the invention provides a method of identifying a product that binds to Brn-3a comprising providing Brn-3a, or a mimic of Brn-3a that can bind to a Brn-3a specific antibody, to a candidate substance and determining whether the candidate substance binds Bra-3a or the mimic of Brn-3a.
  • Brn-3 a for use in the method can be obtained by known techniques.
  • the nucleotide sequence encoding Brn-3a is provided herein as SEQ ID NOT . This sequence information can be used by the skilled man to produce Brn-3a protein using routine methods.
  • the Brn-3a or the mimic is typically (i) human Brn-3 a; (ii) a homologue of (i); or (iii) a fragment of (i) or (ii).
  • the mimic may, however, be a mimic which is not derived from Brn-3a.
  • Such a mimic may have been designed (e.g. computationally) to resemble Brn-3a in its binding characteristics and/or may have been selected (e.g from a library of substances) based on its ability to bind agents which bind Brn-3 a.
  • Brn-3a or the mimic is generally in a suitable buffer, which includes any suitable biological buffer that can provide buffering capability at a pH conducive to the reaction requirements of the Brn-3 a.
  • the Brn-3 a may be in conditions, including temperatures, which are similar to intracellular conditions.
  • the Brn-3a or mimic may be inside a cell or outside a cell.
  • the cell may be the cell in which the Brn-3a naturally occurs, or a cell in which the Brn- 3 a or mimic is expressed recombinantly.
  • the cell may be treated with agents which permeabilise the cell surface allowing test substances to enter the cell more readily.
  • the Brn-3 a or mimic may be in the form of an extract from such cells.
  • Binding assays Methods which determine whether a candidate substance is able to bind the
  • Brn-3a may comprise providing Brn-3a or the mimic to a candidate substance and determining whether binding occurs, for example by measuring the amount of the candidate substance which binds Brn-3 a.
  • the binding may be determined by measuring a characteristic of the Brn-3 a that changes upon binding, such as spectroscopic changes.
  • the assay format may be a 'band shift' system. This involves determining whether a test candidate advances or retards Brn-3 a on gel electrophoresis relative to Brn-3a in the absence of the compound.
  • the method may be a competitive binding method. This determines whether the candidate is able to inhibit the binding of Brn-3 a to an agent which is known to bind to the Brn-3a, such as an antibody specific for Brn-3a. In such a method the candidate substance may inhibit the binding of the agent to Brn- 3a either by the candidate substance binding Brn-3a or by the candidate substance binding the agent.
  • a competitive inhibition method can identify not only substances that bind Brn-3 a, but also substances that act as mimics of Brn-3 a.
  • the competitive binding method may comprise
  • the amount of the labelled reference compound bound to the Brn-3 a may be measured directly or indirectly.
  • a direct measurement may be carried out by removing assay mixture containing the unbound labelled reference compound and measuring the amount of label that is in the product fraction.
  • the amount of labelled reference compound bound to the product could be determined indirectly by measuring the amount of label remaining in the assay solution after removal of the product fraction, which will be inversely related to the amount that has bound to the product.
  • the Brn-3 a may be immobilised on a solid support or may be in solution.
  • immobilised product has the advantage that, after the binding reaction is complete, the product/labelled reference compound complex may be separated from the labelled reference compound that remains in solution by simply removing the solution away from the solid support. If, on the other hand, the product is not immobilised during the assay but rather is in solution, then it will generally be necessary to devise a means for separating the product/labelled reference compound complex from the uncomplexed reference compound before measuring the amount of label. Such separation could be achieved, for example, by precipitating the product using an antibody to the product or by using a non-specific precipitation technique.
  • the different types of assays mentioned above can be used to measure binding between any two substances mentioned herein.
  • a product that inhibits Brn-3a expression and/or activity is one which produces a measurable reduction in Brn-3 a expression and/or activity in the methods described above.
  • Preferred products are those which inhibit Brn-3 a 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 product of l ⁇ g ml "1 , lO ⁇ g ml"', lOO ⁇ g ml "1 , 500 ⁇ g ml " ', lmg ml" 1 ' lOmg ml "1 or lOOmg ml "1 .
  • 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 preferred.
  • the inhibitor may or may not be toxic towards humans or animals.
  • Candidate substances which show activity in assays such as those described herein can then be further tested, for example on malignant cell or in animals with cancer.
  • Products of the invention may be present in a substantially isolated form. It will be understood that the product may be mixed with carriers or diluents which will not interfere with the intended purpose of the product and still be regarded as substantially isolated.
  • a product of the invention may also be in a substantially purified form, in which case it will generally comprise more than 90%, e.g. 95%, 98%) or 99%> of the polypeptide, polynucleotide or dry mass of the preparation.
  • Suitable labels for use in the methods or assays described herein include radioisotopes, e.g. 125 1, 35 S, 32 P enzymes, antibodies, polynucleotides and polypeptides such as biotin.
  • Antibodies to any of the substances discussed herein can be produced by use of the following methods.
  • An antibody to the substance may be produced by raising antibody in a host animal against the whole substance or an antigenic epitope thereof (hereinafter "the immunogen").
  • the immunogen an antigenic epitope thereof
  • a method for producing a polyclonal antibody comprises immunising a suitable host animal, for example an experimental animal, with the immunogen and isolating immunoglobulins from the serum.
  • the animal may therefore be inoculated with the immunogen, blood subsequently removed from the animal and the IgG fraction purified.
  • a method for producing a monoclonal antibody comprises immortalising cells which produce the desired antibody.
  • Hybridoma cells may be produced by fusing spleen cells from an inoculated experimental animal with tumour cells (Kohler and Milstein, Nature 256, 495-497, 1975).
  • An immortalized cell producing the desired antibody may be selected by a conventional procedure.
  • the hybridomas may be grown in culture or injected intraperitoneally for formation of ascites fluid or into the blood stream of an allogenic host or immunocompromised host.
  • Human antibody may be prepared by in vitro immunisation of human lymphocytes, followed by transformation of the lymphocytes with Epstein-Barr virus.
  • the experimental animal is suitably a goat, rabbit, rat or mouse.
  • the immunogen may be administered as a conjugate in which the immunogen is coupled, for example via a side chain of one of the amino acid residues, to a suitable carrier.
  • the carrier molecule is typically a physiologically acceptable carrier.
  • the antibody obtained may be isolated and, if desired, purified.
  • a polynucleotide or polypeptide which is homologous to another polynucleotide or polypeptide is at least 70% homologous to the polynucleotide or polypeptide, preferably at least 80 or 90% and more preferably at least 95%, 97% or 99%o homologous thereto.
  • Such homology may exist over a region of at least 15, preferably at least 30, for instance at least 40, 60 or 100 or more contiguous amino acids.
  • Methods of measuring polynucleotide or polypeptide homology are well known in the art. For example the UWGCG Package provides the BESTFIT program which can be used to calculate homology (Devereux et al (1984) Nucleic Acids Research 12, p387-395).
  • Suitable candidate substances which tested in the above methods include antibody products (for example, monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies and CDR-grafted antibodies) which are specific for Brn-3a or mimics of Brn-3a. Furthermore, combinatorial libraries, defined chemical identities, peptide and peptide mimetics, oligonucleotides and natural product libraries , such as display libraries (e.g. phage display libraries) may also be tested.
  • the candidate substances may be chemical compounds. Batches of the candidate substances may be used in an initial screen of, for example, ten substances per reaction, and the substances of batches which show inhibition tested individually.
  • a mimic of any of the polypeptides which are discussed herein(e.g. the HPV or cellular components) is typically a polypeptide with homology to the original polypeptide.
  • a mimic of any of the polynucleotides discussed herein is typically a polynucleotide with homology to the original polynucleotide.
  • the mimics may be polypeptides or polynucleotides which are not homologous, or can be non-polypeptide or non-polynucleotide substances.
  • a mimic of a substance binds a specific antibody which is able to bind the substance.
  • the mimic typically has a shape, size, flexibility or electronic configuration which is substantially similar to the original substance. It is typically a derivative of the original substance.
  • products of the invention may be expressed from polynucleotides in vivo, particularly in the case of products which are antisense polynucleotides.
  • the product is expressed in a cell from a recombinant replicable vector.
  • a replicable vector comprises a polynucleotide which when transcribed gives rise to product.
  • the product may be provided by delivering such a vector to the cell and allowing transcription from the vector to occur.
  • a vector is understood to be a 'product' of the invention.
  • the polynucleotide giving rise to the product is operably linked to a control sequence which is capable of providing for the transcription of the polynucleotide giving rise to the product.
  • Operably linked' refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner.
  • a control sequence 'operably linked' to a sequence giving rise to the product is ligated in such a way that transcription of the sequence is achieved under conditions compatible with the control sequences.
  • the vector may be for example, a plasmid or virus vector provided with an origin of replication, optionally a promoter for transcription to occur and optionally a regulator of the promoter.
  • the vector may contain one or more selectable marker genes, for example an ampicillin resistance gene in the case of bacterial plasmid or a neomycin resistance gene for a mammalian vector.
  • Vectors may be used in vitro, for example for the production of the product or used to transfect or transform a host cell.
  • the term 'host cell' refers to a cell of any of the cancers discussed above. It may be a malignant or normal cell.
  • the vector may also be adapted to be used in vivo, for example in a method of gene therapy.
  • 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
  • Tissue-specific promoters in particular cervical or epithelial cell specific promoters (for example the Brn-3a involucin or keratin promoters), are especially preferred.
  • Viral promoters may also be used, for example the Moloney murine leukaemia virus long terminal repeat (MMLV LTR), the rous sarcoma virus (RSV) LTR promoter, the SV40 promoter, the human cytomegalovirus (CMV) LE promoter, adenovirus, HSV promoters (such as the HSV IE promoters), or HPV promoters, particularly the HPV upstream regulatory region (URR). All these promoters are readily available in the art.
  • MMLV LTR Moloney murine leukaemia virus long terminal repeat
  • RSV rous sarcoma virus
  • CMV human cytomegalovirus
  • HSV promoters such as the HSV IE promoters
  • HPV promoters particularly the HPV upstream regulatory region (URR). All these promoters are readily available in the art.
  • the vector may further include sequences flanking the polynucleotide giving rise to antisense RNA which comprise sequences homologous to eukaryotic genomic sequences, preferably mammalian genomic sequences, or viral genomic sequences.
  • sequences flanking the polynucleotide giving rise to antisense RNA which comprise sequences homologous to eukaryotic genomic sequences, preferably mammalian genomic sequences, or viral genomic sequences.
  • sequences flanking the polynucleotide giving rise to antisense RNA which comprise sequences homologous to eukaryotic genomic sequences, preferably mammalian genomic sequences, or viral genomic sequences.
  • Suitable viral vectors include herpes simplex viral vectors (for example as disclosed in WO 98/04726 and WO 98/30707) and retroviruses, including lentiviruses, adenoviruses, adeno-associated viruses and HPV viruses (such as HPV-16 or HPV-18). Gene transfer techniques using these viruses are known to those skilled in the art. Retrovirus vectors for example may be used to stably integrate the polynucleotide giving rise to the antisense RNA into the host genome. Replication-defective adenovirus vectors by contrast remain episomal and therefore allow transient expression.
  • the vectors and antisense oligonucleotides of the invention may thus be administered to a human or animal in need of treatment.
  • Cancers which may be treated using the vectors, viral strains, antisense oligonucleotides and compositions of the invention cervical cancers attribute to HPV. The condition of a patient suffering from such a cancer can thus be improved.
  • the antisense oligonucleotides and compositions comprising antisense oligonucleotides of the invention together may be administered by direct injection into the site to be treated, for example cervical tissue.
  • the antisense oligonucleotides are combined with a pharmaceutically acceptable carrier 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, 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.
  • the polynucleotides giving rise to the product of the invention in vivo 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 cationic agents (for example calcium phosphate and DEAE-dextran) and lipofectants (for example lipofectam 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 carrier 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, 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 inco ⁇ orated into cells at an appropriate area.
  • the amount of virus administered is in the range of
  • Such a system may, for example, involve administering the antibiotic tetracycline, to activate gene expression via its effect on the tet repressor/VP 16 fusion protein.
  • tissue-specific promoters will be of assistance in the treatment of disease using the polypeptides, polynucleotide and vectors of the invention. 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 routes of administration and dosages described above are intended only as a guide since a skilled physician will be able to determine readily the optimum route of administration and dosage for any particular patient and condition.
  • Human use Products found to bind, cause a decrease in intracellular levels of, or inhibit the activity of Brn-3a in the screening procedures described above may be used to treat the cancers discussed above.
  • the condition of a patient suffering from such cancer can therefore be improved by administration of such a product.
  • a therapeutically effective amount of such an product may be given to a human patient in need thereof.
  • the formulation of the product for use in preventing or treating the cancer will depend upon factors such as the nature of the substance identified, whether a pharmaceutical or veterinary use is intended, etc.
  • the product is formulated for use with a pharmaceutically acceptable carrier or diluent.
  • a pharmaceutically acceptable carrier or diluent For example it may be formulated for topical, parenteral, intravenous, intramuscular, subcutaneous, transdermal or oral administration. A physician will be able to determine the required route of administration for each particular patient.
  • the pharmaceutical carrier or diluent may be, for example, an isotonic solution.
  • the dose of product may be determined according to various 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.
  • a suitable dose may however be from 0.1 to 100 mg/kg body weight such as 1 to 40 mg/kg body weight. Again, a physician will be able to determine the required route of administration and dosage for any particular patient.
  • Sections were histologically classified in respect of the presence of cervical intraepithelial neoplasia. High grade lesions were those with a diagnosis of CLN2/3 and low grade of CLN1. Normal tissues were also classified. The biopsies were made up of 1) 14 CTN3 lesions (CLN3), 2) 16 normal samples from regions adjacent to CIN3 lesions (N-CIN), and 3) 16 normal samples from individuals with no histologically detectable cervical abnormality
  • cDNA Complementary DNA obtained from human neuroblastoma cell lines expressing Brn-3a and Brn-3b was used as positive controls for RT-PCR experiments Water only or human fibroblast mRNA (which does not express Brn-3a or Brn-3b) was used as a negative control Plasmids with human Brn-3a and Brn-3b cDNA were used as positive controls for polymerase chain reaction (PCR) experiments HPV- 16 DNA was used as positive control for subsequent HPV PCR procedures, with water or DNA or RNA from an HPV-negative neuroblast
  • RNA and DNA extractions from human cervical biopsies and Reverse Transcriptase (RT)-PCR RNA was prepared using the guanidinium isothiocyanate method (2)
  • the samples of about 33mg were homogenised in 250 ⁇ l denaturation solution containing 4M guanidinium thiocyanate, 25mM sodium citrate, pH7, 0 IM 2-mercaptoethanol, 0 5% N-Laurolsarcosine DNA was extracted using guanidinium isothiocyanate as previously described
  • Resultant RNA and DNA were respectively treated with 250mg/ml DNase and RNase About 0 1 ⁇ g of the resultant RNA from each sample was used as a template for cDNA synthesis
  • the synthesised cDNA was used in RT-PCR assays as previously described (4, 5) using the following oligonucleotide primers - Brn-3 a 5'GTCGACATGGACTCGGACACG-3', 3'- ACGGTGAA
  • Samples for western blot protein assay were made up of 1 1 CLN3 lesions, 10 normal samples from adjacent regions and 13 normal samples from women with no histologically detectable abnormality. They were resuspended in sample buffer containing 2.3% sodium dodecyl sulphate. 0.0625M Tris HCL, pH7.9, 10% glycerol, 5% ⁇ -mercaptoethanol, and bromphenol blue. Samples were sonicated for 10 seconds and then boiled for 2min. The samples had equal protein content, as determined by the method of Bradford (9) and were split in two and run on two sides of the same SDS-polyacrylamide gel.
  • Values are in arbitary densitometric units equalised for the level of cyclophilin mRNA in each sample.
  • the expression vector pLTRpoly(ATCC) containing the full length of the class IV POU domain transcription factors Brn-3a and Brn-3b under the Moloney murine leukaemia virus promoter (MoMuLV) have previously been described (14).
  • the antisense Brn-3a construct was cloned within the pJ5 vector polylinker, under the control of the glucocorticoid-inducible mouse mammary tumour virus promoter
  • SiHa (ATCC) and C33 (ATCC) cell lines were grown in Minimum essential medium (Eagle) with Earle's BSS, supplemented with 10% fetal bovine serum, 0. ImM non-essential amino acids and 1.OmM sodium pyruvate.
  • the Brn-3 expression vectors were cotransfected with pCi-neo (Promega) neomycin resistant vector into both cell lines by calcium phosphate-mediated transfection method (16).
  • the gel was blotted onto membrane (Amersham) and the membrane was blocked for 2 hours with 10% Marvel (fat-free milk) and incubated with 1 500 HPV- 16-E6 antibody (Santa Cruz) for 16 hours overnight at 4°C, washed 5 times with
  • the two cell lines expressing the antisense construct showed a clear decrease in the level of E6 gene expression which was greatest in cell line 5 paralleling the greater reduction in Brn- 3a levels in this cell line compared to the antisense cell line 3.
  • no HPV gene expression was detected in any of the cell lines derived from C-33 cells which are not transformed with HPV.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Immunology (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Pathology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Medicinal Chemistry (AREA)
  • Hospice & Palliative Care (AREA)
  • Oncology (AREA)
  • Virology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

Cette invention concerne une substance qui lie le facteur de transcription humaine Brn-3a, provoque une diminution des niveaux intracellulaires de ce facteur ou en inhibe l'activité, et qui convient pour le traitement, la prévention ou le diagnostic du cancer du col de l'utérus attribuable au virus HPV. L'invention concerne également des méthodes d'identification de cette substance. Cette substance peut également être utilisée à des fins de dépistage chez des individus exposés à des risques de cancer du col de l'utérus.
PCT/GB1999/004116 1998-12-07 1999-12-07 Traitement du cancer du col de l'uterus WO2000034466A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP99959533A EP1151094A1 (fr) 1998-12-07 1999-12-07 Traitement du cancer du col de l'uterus

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
GBGB9826888.1A GB9826888D0 (en) 1998-12-07 1998-12-07 Cervical cancer treatment
GB9826888.1 1998-12-07
US28221099A 1999-03-31 1999-03-31
US09/282,210 1999-03-31

Publications (1)

Publication Number Publication Date
WO2000034466A1 true WO2000034466A1 (fr) 2000-06-15

Family

ID=26314798

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB1999/004116 WO2000034466A1 (fr) 1998-12-07 1999-12-07 Traitement du cancer du col de l'uterus

Country Status (2)

Country Link
EP (1) EP1151094A1 (fr)
WO (1) WO2000034466A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003036300A2 (fr) * 2001-10-26 2003-05-01 University College London Diagnostic du cancer du col de l'uterus
WO2005090608A2 (fr) * 2004-03-05 2005-09-29 Advandx, Inc. Sondes haute affinite pour analyse de l'expression du papillomavirus humain
US7704965B2 (en) 2002-06-26 2010-04-27 The Penn State Research Foundation Methods and materials for treating human papillomavirus infections
US7736654B2 (en) 2001-04-10 2010-06-15 Agensys, Inc. Nucleic acids and corresponding proteins useful in the detection and treatment of various cancers

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999005272A1 (fr) * 1997-07-25 1999-02-04 Neurovex Limited Utilisation du facteur de transcription brn-3a

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999005272A1 (fr) * 1997-07-25 1999-02-04 Neurovex Limited Utilisation du facteur de transcription brn-3a

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
DATABASE BIOSIS [online] BIOSCIENCES INFORMATION SERVICE, PHILADELPHIA, PA, US; 1994, MORRIS PETER J ET AL: "The opposite and antagonistic effects of the closely related POU family transcription factors Brn-3a and Brn-3b on the activity of a target promoter are dependent on differences in the POU domain.", XP002135724, Database accession no. PREV199497544582 *
DATABASE BIOSIS [online] BIOSCIENCES INFORMATION SERVICE, PHILADELPHIA, PA, US; 1997, LATCHMAN DAVID S: "Regulation of neuroblastoma growth and differentiation by the POU family transcription factors Brn-3a and Brn-3b (review).", XP002135725, Database accession no. PREV199799597457 *
INTERNATIONAL JOURNAL OF ONCOLOGY 1997, vol. 10, no. 6, 1997, pages 1133 - 1139, ISSN: 1019-6439 *
MOLECULAR AND CELLULAR BIOLOGY 1994, vol. 14, no. 10, 1994, pages 6907 - 6914, ISSN: 0270-7306 *
NDISANG DANIEL ET AL: "The Brn-3a transcription factor plays a critical role in regulating human papilloma virus gene expression and determining the growth characteristics of cervical cancer cells.", JOURNAL OF BIOLOGICAL CHEMISTRY OCT. 1, 1999, vol. 274, no. 40, 1 October 1999 (1999-10-01), pages 28521 - 28527, XP002135723, ISSN: 0021-9258 *
NDISDANG DANIEL ET AL: "The HPV-activating cellular transcription factor Brn-3a is overepressed in CIN3 cervical lesions.", JOURNAL OF CLINICAL INVESTIGATION APRIL 15, 1998, vol. 101, no. 8, 15 April 1998 (1998-04-15), pages 1687 - 1692, XP000867063, ISSN: 0021-9738 *
SMITH ET AL: "The Brn-3a transcription factor induces neuronal proceses outgrowth and the coordinate expression of genes encoding synaptic proteins", MOLECULAR AND CELLULAR BIOLOGY,US,WASHINGTON, DC, vol. 17, no. 1, 1 January 1997 (1997-01-01), pages 345 - 354, XP002087059, ISSN: 0270-7306 *
THEIL THOMAS ET AL: "Short isoform of POU factor Brn-3b can form a heterodimer with Brn-3a that is inactive for octamer motif binding.", JOURNAL OF BIOLOGICAL CHEMISTRY 1995, vol. 270, no. 52, 1995, pages 30958 - 30964, XP002135722, ISSN: 0021-9258 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7736654B2 (en) 2001-04-10 2010-06-15 Agensys, Inc. Nucleic acids and corresponding proteins useful in the detection and treatment of various cancers
WO2003036300A2 (fr) * 2001-10-26 2003-05-01 University College London Diagnostic du cancer du col de l'uterus
WO2003036300A3 (fr) * 2001-10-26 2003-10-30 Univ London Diagnostic du cancer du col de l'uterus
US7704965B2 (en) 2002-06-26 2010-04-27 The Penn State Research Foundation Methods and materials for treating human papillomavirus infections
WO2005090608A2 (fr) * 2004-03-05 2005-09-29 Advandx, Inc. Sondes haute affinite pour analyse de l'expression du papillomavirus humain
WO2005090608A3 (fr) * 2004-03-05 2006-10-12 Advandx Inc Sondes haute affinite pour analyse de l'expression du papillomavirus humain

Also Published As

Publication number Publication date
EP1151094A1 (fr) 2001-11-07

Similar Documents

Publication Publication Date Title
CA2273821C (fr) Detection et modulation d'inhibiteurs de proteines d'apoptose (iap) et de polypeptides anti-apoptotiques apparentes (miap) permettant d'effectuer le diagnostic et le traitement demaladies proliferatives
EP1009753B1 (fr) Genes regules au niveau de l'hypoxemie
CA2315279A1 (fr) 14-3-3.sigma. bloque le cycle cellulaire
JP2011244827A (ja) 染色体３ｐ２１．３遺伝子は腫瘍サプレッサーである
JP2002512788A (ja) 分化をモジュレートする方法
JP2002525081A (ja) 低酸素により調節される遺伝子転写に特徴的な配列
JP4005135B2 (ja) 神経アポトーシス抑制性蛋白質（ｎａｉｐ）の使用
US20080182250A1 (en) Method for detecting multiple myeloma and method for inhibiting the same
WO2000034466A1 (fr) Traitement du cancer du col de l'uterus
US7776519B2 (en) Use of the slug gene, or of the transcription or expression products thereof in the detection and/or treatment of cancerous cells
KR20090060212A (ko) 암의 검출 및 치료를 위한 조성물 및 방법
US6930175B1 (en) Gene family with transformation modulating activity
EP1045926B1 (fr) Famille de genes possedant une activite de modulation de la transformation
JP2002525269A (ja) Ｂｒｎ−３ｂの阻害因子ならびに乳癌および卵巣癌の治療のためのその使用
US20080096210A1 (en) Cancer suppressing agent
EP1613769B1 (fr) Gene induit par l'insuline utilise comme cible therapeutique dans le diabete
JP4723133B2 (ja) 新規ヒトｂｍｃｃ１遺伝子
US7598077B2 (en) Compositions and methods for enhancing differential expression
WO2008075713A1 (fr) Marqueur pour le diagnostic du cancer et molécule cible pour une thérapie
AU2003204082B2 (en) Gene family with transformation modulating activity
US20050208553A1 (en) Methods of detecting and treating microsatellite-instability positive tumors using RIZ
EP1146909A1 (fr) Traitement du cancer par restauration de la fonction pp32
AU2005200892A1 (en) Method of treating cancer by restoration of PP32 function
US20030032606A1 (en) Methods of detecting and treating microsatellite-instability positive tumors using RIZ
US20080311090A1 (en) Methods for inhibition of proliferative disease, including hepatocellular carcinoma

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): JP

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
WWE Wipo information: entry into national phase

Ref document number: 1999959533

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 1999959533

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 1999959533

Country of ref document: EP