WO2001075099A1 - Molecule d'acide nucleique codant une proteine uterine pouvant etre induite par un agoniste d'oestrogene - Google Patents

Molecule d'acide nucleique codant une proteine uterine pouvant etre induite par un agoniste d'oestrogene Download PDF

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WO2001075099A1
WO2001075099A1 PCT/AU2001/000379 AU0100379W WO0175099A1 WO 2001075099 A1 WO2001075099 A1 WO 2001075099A1 AU 0100379 W AU0100379 W AU 0100379W WO 0175099 A1 WO0175099 A1 WO 0175099A1
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sequence
seq
nucleic acid
set forth
nucleotide sequence
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PCT/AU2001/000379
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English (en)
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The Hung Huynh
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National Cancer Centre Of Singapore Pte Ltd
Hughes, Edward, John, Langfo
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Priority claimed from AUPQ9471A external-priority patent/AUPQ947100A0/en
Application filed by National Cancer Centre Of Singapore Pte Ltd, Hughes, Edward, John, Langfo filed Critical National Cancer Centre Of Singapore Pte Ltd
Priority to AU43962/01A priority Critical patent/AU4396201A/en
Publication of WO2001075099A1 publication Critical patent/WO2001075099A1/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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants

Definitions

  • the present invention relates generally to a nucleic acid molecule comprising a nucleotide sequence corresponding to an estrogen agonist-inducible genetic sequence in cells derived from the uterus.
  • the nucleic acid molecule of the present invention encodes a protein which is proposed to mediate the effects of estrogenic hormones and other estrogen agonists in inducing epithelial proliferation and differentiation in uterine and ovarian tissues.
  • the present invention provides, therefore, a means of monitoring uterine and ovarian growth and development as well as the level of estrogenic activity in tissues including cancer tissue by, for example, measuring expression of the subject nucleic acid molecule and/or detecting the level of the estrogen agonist-mediating polypeptide encoded by said nucleic acid molecule.
  • the present invention further provides compositions comprising the subject polypeptide or its derivatives, homologs, analogs or agonists.
  • the present invention further contemplates a method of modulating the growth of uterine and/or ovarian tissue including uterus- or ovary-derived cancer tissue by the administration of an agonist or antagonist of expression of the subject nucleic acid molecule or activity of the polypeptide encoded by same.
  • the instant nucleic acid molecule also provides directly or indirectly via its expression product a means for diagnosing, detecting or otherwise monitoring uterine cancers and/or ovarian cancers.
  • the present invention further provides a promoter and/or other regulatory regions associated with the expression of the subject nucleic acid molecule.
  • the female genital tract including the uterus, exhibits considerable control over the ability of a conceptus to develop (1, 2).
  • the uterus expresses and secretes a number of growth factors (3-8) and other regulatory polypeptides (7, 9) in response to ovarian steroid hormones. These polypeptides are thought to play a part in directing or limiting the growth and development of the uterus.
  • Estrogens promote the growth, differentiation and remodelling of the uterus during the estrus cycle and pregnancy (10-12). They modulate the expression of genes involved in the regulation of cell growth and differentiation including EGF, IGF-I and their receptors (5, 13-20).
  • Preovulatory ovarian estrogen secretion is important for uterine cellular proliferation and epithelial differentiation during early stages of pregnancy (21).
  • the CUB domain is an extracellular domain of approximately 110 residues which is found in functionally diverse (mostly developmental) proteins. Proteins that contain the CUB domain include mammalian complement subcomponents Cls/CIR (22, 23); hamster serine protease, Casp (24, 25); and mammalian complement-activating component of Ra-reactive factor (RARF) (26); vertebrate bone morphogenic protein 1 (BMP-1) (27-31); neuropilin (A5 antigen) (32-39); mammalian hyaluronate-binding protein TSG-6, a serum- and growth factor-induced protein possibly involved in cell-cell and cell-matrix interactions during inflammation and tumorigenesis (40); and mammalian spermadhesins (24, 41, 42).
  • mammalian complement subcomponents Cls/CIR 22, 23
  • hamster serine protease Casp
  • RARF mammalian complement-activating component of Ra-reactive factor
  • Bovine acidic seminal fluid contains a CUB domain and belongs to the spermadhesin family. It functions both as a mitogen and growth factor in vitro (43) and a stimulator of progesterone secretion in cultured ovarian cells (43).
  • the anti-estrogen drug tamoxifen improves the survival of women with breast cancer and has proved to be clinically useful for the treatment of metastic estrogen receptor-positive tumours (44).
  • long-term administration of tamoxifen has been reported to be associated with an increased risk of endometrial cancer in post-menopausal women (45).
  • Tamoxifen causes endometrial thickening in some postmenopausal women.
  • Over 40% of women on tamoxifen had an endometrium >8 mm thick compared with only 5% of control women of placebo (46). It has been suggested that the estrogenic effect of tamoxifen on the atrophic postmenopausal endometrium causes hyperplasia that may progress to atypia and cancer in a manner similar to that seen with estrogen replacement therapy.
  • the inventors sought to identify tamoxifen- and estradiol-regulated genes in the uterus using differential display to examine the transcript expression profile of the ovariectomized uterus under conditions of tamoxifen supplementation.
  • a novel tamoxifen-inducible nucleic acid molecule has been isolated.
  • the instant nucleic acid molecule is proposed to be important in normal and neoplastic uterine and ovarian growth. It is also proposed that the nucleic acid molecule and/or its expression product will serve as a biomarker for uterine and ovarian cancer and aid in their diagnosis and treatment.
  • SEQ ID NO: Nucleotide and amino acid sequences are referred to by a sequence identifier number (SEQ ID NO:).
  • the SEQ ID NOs: correspond numerically to the sequence identifiers ⁇ 400>1, ⁇ 400>2, etc.
  • a sequence listing is provided after the claims.
  • One aspect of the present invention provides a nucleic acid molecule comprising a sequence of nucleotides corresponding to a uterine estrogen agonist-inducible genetic sequence.
  • nucleic acid molecule comprising a sequence of nucleotides corresponding to a uterine estrogen agonist-inducible genetic sequence which is expressed in estrogen agonist treated ovariectomized uterus cells or tissue but substantially not or in very low amounts in non-estrogen agonist treated ovariectomized uterine tissue.
  • a further aspect of the present invention provides a nucleic acid molecule comprising a sequence of nucleotides substantially as set forth in SEQ ID NO:l or a nucleotide sequence having at least 50% similarity thereto or a nucleotide sequence capable of hybridizing to SEQ ID NO: 1 or a complementary form thereof under low stringency conditions.
  • Still another aspect of the present invention is directed to a nucleic acid molecule a comprising a sequence of nucleotides substantially as set forth in SEQ ID NO:l or a nucleotide sequence having at least 50% similarity thereto or a nucleotide sequence capable of hybridizing to SEQ ID NO:l or a complementary form thereof under low stringency conditions wherein said nucleic acid molecule corresponds to a uterine estrogen agonist-inducible genetic sequence.
  • Still a further aspect of the present invention provides a nucleic acid molecule comprising a sequence of nucleotides substantially as set forth in SEQ ID NO:3 or a nucleotide sequence having at least 50% similarity thereto or a nucleotide sequence capable of hybridizing to SEQ ID NO: 3 or a complementary form thereof under low stringency conditions.
  • nucleic acid molecule a comprising a sequence of nucleotides substantially as set forth in SEQ ID NO:3 or a nucleotide sequence having at least 50% similarity thereto or a nucleotide sequence capable of hybridizing to SEQ ID NO:3 or a complementary form thereof under low stringency conditions wherein said nucleic acid molecule corresponds to a uterine estrogen agonist-inducible genetic sequence.
  • Yet another aspect of the present invention relates to a genetic construct comprising a nucleotide sequence corresponding to a uterine estrogen agonist-inducible genetic sequence.
  • Still another aspect of the present invention contemplates a method for producing a uterine estrogen agonist-mediating polypeptide said method comprising introducing into a cell a nucleic acid molecule comprising a nucleotide sequence corresponding to a uterine estrogen agonist-inducible genetic sequence and subjecting said cell to conditions sufficient to permit expression of the nucleotide sequence and then recovering the polypeptide or membrane portions comprising same.
  • Another aspect of the present invention provides a polypeptide comprising a sequence of amino acids corresponding to a uterine estrogen agonist-mediating polypeptide.
  • a further aspect of the present invention is directed to a polypeptide comprising sequence of amino acids encoded by the nucleotide sequence substantially as set forth in SEQ ID NO:l or a nucleotide sequence having at least 50% similarity thereto or a nucleotide sequence capable of hybridizing SEQ ID NO:l under low stringency conditions.
  • Still another aspect of the present invention provides a polypeptide comprising a sequence of amino acids substantially as set forth in SEQ ID NO:2 or an amino acid sequence having at least about 50% ⁇ similarity thereto.
  • the present invention provides a polypeptide comprising a sequence of amino acids substantially as set forth in SEQ ID NO:2 or an amino acid sequence having at least about 50% similarity thereto and which polypeptide corresponds to a uterine estrogen agonist-mediating polypeptide or a mutant, derivative, homologue or analogue of said polypeptide.
  • Yet another aspect of the present invention is directed to a polypeptide comprising sequence of amino acids encoded by the nucleotide sequence substantially as set forth in SEQ ID NO: 3 or a nucleotide sequence having at least 50% similarity thereto or a nucleotide sequence capable of hybridizing SEQ ID NO: 3 under low stringency conditions.
  • Still another aspect of the present invention provides a polypeptide comprising a sequence of amino acids substantially as set forth in SEQ ID NO:4 or an amino acid sequence having at least about 50% similarity thereto.
  • the present invention provides a polypeptide comprising a sequence of amino acids substantially as set forth in SEQ ID NO:4 or an amino acid sequence having at least about 50% similarity thereto and which polypeptide corresponds to a uterine estrogen agonist-mediating polypeptide or a mutant, derivative, homologue or analogue of said polypeptide.
  • Figure 1 is a photographic representation showing differentially expressed mRNA bands in uterine tissues of control ONX and ONX-tamoxifen-treated rats.
  • Total R ⁇ A was isolated from uteri of ONX and ONX-tamoxifen-treated rats was subjected to differential display.
  • the band representing mR ⁇ A that was induced during tamoxifen treatment is marked by an arrowhead.
  • Northern blot analysis of total RNA from uteri of ONX and ONX-tamoxifen- treated rats was used to confirmed the presence of a differentially expressed mR ⁇ A in the ONX-tamoxifen-treated uteri. Blots were hybridized with a 32 P-labelled cD ⁇ A fragment that was isolated from the above differential gel (A) or GAPDH cD ⁇ A (B).
  • FIG. 2 is a photographic representation showing Northern blot analysis of UO-44 gene expression in female adult rat tissues.
  • Total RNA derived from various tissues of an 80-day old female rat was subjected to Northern blot analysis. Blots were hybridized with rat UO-44 (A) and GAPDH (B) cDNAs.
  • Tissues are: Mg: mammary gland; Fa: abdominal fat; Mu: red muscle; Ov: ovary; He: heart; Lu: lung; Li: liver; Sto: stomach; Int: small intestine; Spl: spleen; Pi: pituitary; Br: brain; Ki: kidney; and Ut: uterus.
  • Figure 3 is a representation showing ovarian-steroid dependent UO-44 gene expression.
  • Female rats were underwent ovariectomy. The uteri were collected at the indicated times after ovariectomy. Total RNA derived from uteri was subjected to Northern blotting. Blots were hybridised with GAPDH (A, D) and rat UO-44 (B, E) cDNAs. Time-dependent tamoxifen- induced UO-44 gene expression in uteri of ONX-rats is shown in (E). Densitometric scanning of the UO-44 band is shown in (C, F). Bars with different letters are significantly different from one another at (p ⁇ .01). Data are expressed as the mean ⁇ SEM.
  • FIG 4 is a representation showing effects of tamoxifen treatment on UO-44 expression in the uteri of ONX rats.
  • Ovariectomized (OVX) rats were treated with indicated amount of tamoxifen (TAM), 1.2 ⁇ g/day estradiol (E 2 ) or 5 mg tamoxifen plus 1 mg ICI 182780 (T M + ICI) for 3 weeks.
  • Total R ⁇ A derived from uteri was subjected to Northern blotting. Blots were hybridized with GAPDH (A) and rat UO-44 (B) cDNAs. Densitometric scanning of the UO-44 band (C) and the effect of each treatment on uterine weight (D) are shown.
  • Uteri of ovary intact (I) serves as a positive control. Bars with different letters are significantly different from one another at (pO.Ol). Data are expressed as the mean + SEM.
  • Figure 5 is a representation showing effects of tamoxifen and ICI 182780 on UO-44 gene expression and uterine weight of ovary-intact rats.
  • Ovary-intact rats were treated with indicated concentrations of tamoxifen (TAM, A), and ICI 182780 (ICI, B) for 3 weeks.
  • Total RNA derived from uteri was subjected to Northern blot analysis. Blots were hybridized with GAPDH and rat UO-44 cDNAs. Densitometric scanning of the UO-44 band and the effect of the treatments on uterine weight are shown. Bars with different letters are significantly different from one another at (p ⁇ .01). Data are expressed as the mean ⁇ SEM.
  • Figure 6 is a representation showing effects of growth hormone on UO-44 gene expression in the uterus of hypophysectomized rats. Hypophysectomized rats were treated with vehicle, or indicated concentration of human growth hormone (GH) per gram body weight for 3 weeks. Total RNA derived from uteri was analyzed by Northern blotting. Blots were hybridized with rat UO-44 or GAPDH cDNAs (A). Densitometric scanning of the UO-44 band (B) and the effect of GH on uterine weight (C) are shown. Bars with different letters are significantly different from one another at (pO.Ol). Data are expressed as the mean ⁇ SEM.
  • GH human growth hormone
  • Figure 7 is a photographic representation showing effects of tamoxifen, ICI 182780, growth hormone, and estradiol on UO-44 gene expression in the ovaries of hypophysectomized rats.
  • Hypophysectomized rats were treated with vehicle (C), 1 ⁇ g human growth hormone per gram body weight (GH), 1.0 mg ICI 182780 per kg BW per week (ICI), 1.2 ⁇ g 17- ⁇ estradiol per day (E 2 ) and 5 mg tamoxifen per kg BW per day (TAM).
  • Total RNA derived from ovaries was analyzed by Northern blotting. Blots were hybridized with rat UO-44 (A) or GAPDH cDNAs (B). Hypophysectomy had no effect on UO-44 gene expression in the ovaries
  • Figure 8 is a photographic representation showing detection of UO-44 mRNA in the tamoxifen-treated uteri. In situ hybridization with antisense RNA probe for UO-44 gene expression in the uteri of ONX-tamoxifen-treated uteri at low (A) and high magnification (B). (C) Sense control UO-44 probe showing no background staining in uterus of tamoxifen- treated rats. In situ hybridization with antisense R ⁇ A probe for UO-44 gene expression in the ONX-tamoxifen-ICI-treated uteri (D). UO-44 mR ⁇ A was localized in the luminal secretory epithelial cells and glandular epithelial cells. ICI abolished tamoxifen-induced UO-44 expression in the uteri of ONX rats.
  • Figure 9 is a photographic representation showing in situ hybridization with antisense R ⁇ A probe for UO-44 expression in rat ovaries.
  • Low- (A) and high- (B) magnification showing the non-uniform UO-44 mR ⁇ A distribution in the follicles and granulosa cells. Abundant levels of UO-44 expression were detected in the granulosa cells of medium size follicles. Moderate levels UO-44 mR ⁇ A were detected in granulosa cells of large and small follicles.
  • C A sense control UO-44 probe showed no background staining in the ovarian tissue.
  • D ICI 182780- treated ovary hybridized with anti-sense UO-44 showed very faint staining signal.
  • FIG. 10 is a photograpic representation showing Subcellular localization of UO-44 protein.
  • Human breast cancer MCF-7 cells were transfected with mammalian expression vector containing full length UO-44 cD ⁇ A (UO-44 pcD ⁇ A3.1/His) or control ⁇ cDNA3.1/His vector as described in Example 6.
  • Plasma membrane-enriched subcellular fractions and cytosolic proteins were isolated and Western blot analysis was performed as described in Example 6. Blots were incubated with mouse anti 6-Histidine antibody and horseradish peroxidase-conjugated donkey anti-mouse secondary antibody. Blots were visualized with a chemiluminescent detection system. Molecular weights of irnmunoreactive bands are shown.
  • the present invention is predicted in part on the identification, cloning and characterization of a nucleic acid molecule in the form of a genetic sequence from cells of uterine origin, the expression of which, is induced by a group of molecules referred to herein as "estrogen agonists".
  • Cells of uterine origin are deemed herein to include ovarian cells.
  • an "estrogen agonist” is deemed to include estrogen and its functional derivatives, homologs and analogs as well as estradiol, tamoxifen, diethylstilbestrol and growth hormone. Tamoxifen acts as an anti-estrogen in breast tissue but exhibits estrogen-like activity in the uterus (47). Expression of the genetic sequence, however, is not induced by the anti-estrogen ICI 182780 which is a 7 alpha-alkylsulf ⁇ nyl analogue of estradiol, ICI 182780 is an estrogen antagonist.
  • the subject genetic sequence and more particularly its expression product, is involved in or otherwise associated with uterine growth mediated by an estrogen agonist.
  • uterine growth includes ovarian growth.
  • the subject genetic sequence is referred to herein as a "uterine estrogen agonist-inducible genetic sequence”.
  • the corresponding expression product is referred to herein as a "uterine estrogen agonist-mediating polypeptide”.
  • one aspect of the present invention provides a nucleic acid molecule comprising a sequence of nucleotides corresponding to a uterine estrogen agonist-inducible genetic sequence.
  • an estrogen agonist includes but is not limited to estrogen, estradiol, tamoxifen, diethylstilbestrol and growth hormone.
  • nucleic acid molecule may, therefore, be DNA or RNA such as but not limited to single or double stranded DNA or RNA.
  • the term "gene” is used in its broadest sense and includes cDNA corresponding to the exons of a gene. Accordingly, reference herein to a “gene” is to be taken to include:-
  • a classical genomic gene consisting of transcriptional and/or translational regulatory sequences and/or a coding region and or non-translated sequences (i.e. introns, 5'- and 3'- untranslated sequences); or
  • gene is also used to describe synthetic or fusion molecules encoding all or part of an expression product, hi particular embodiments, the term “nucleic acid molecule”, “genetic sequence” and “gene” may be used interchangeably.
  • corresponding means that the nucleotide sequences of the nucleic acid molecule has complete or partial identity or at least similarity with the uterus estrogen agonist-inducible genetic sequence in the genome or corresponds to the coding regions of this genetic sequence including any or all of its splice variants and polymorphisms. Where the corresponding sequences are not identical, it may differ by one or more nucleotide substitutions, additions and/or deletions whether or not such charges are degenerate.
  • the uterus estrogen agonist-inducible genetic sequence is conveniently identified following differential display.
  • nucleic acid molecule comprising a sequence of nucleotides corresponding to a uterine estrogen agonist-inducible genetic sequence which is expressed in estrogen agonist treated ovariectomized uterus cells or tissue but substantially not or in very low amounts in non-estrogen agonist treated ovariectomized uterine tissue.
  • low amounts includes basal levels of expression product or at least 20% less than the amount in estrogen agonist treated ovariectomized uterus cells.
  • low amounts also includes not detecting any activity using the procedures currently available.
  • the nucleic acid molecule is referred to herein as "UO-44".
  • the murine form of UO-44 such as from rat (i.e. rUO-44) comprises the nucleotide sequence substantially as set forth in SEQ ID NO:l.
  • the human form of UO-44 (hUO-44) comprises the nucleotide sequence set forth in SEQ ID NO:3.
  • the terms "UO- 44", “rUO-44", “hUO-44” or equivalent forms also encompass derivatives, homologs and analogs of UO-44.
  • nucleic acid molecule comprising a sequence of nucleotides substantially as set forth in SEQ ID NO:l or a nucleotide sequence having at least 50% similarity thereto or a nucleotide sequence capable of hybridizing to SEQ ID NO:l or a complementary form thereof under low stringency conditions.
  • the present invention is directed to a nucleic acid molecule comprising a sequence of nucleotides substantially as set forth in SEQ ID NO:l or a nucleotide sequence having at least 50% similarity thereto or a nucleotide sequence capable of hybridizing to SEQ H) NO:l or a complementary form thereof under low stringency conditions wherein said nucleic acid molecule corresponds to a uterine estrogen agonist-inducible genetic sequence.
  • the present invention provides a nucleic acid molecule comprising a sequence of nucleotides substantially as set forth in SEQ ID NO:3 or a nucleotide sequence having at least 50% similarity thereto or a nucleotide sequence capable of hybridizing to SEQ ID NO:3 or a complementary form thereof under low stringency conditions.
  • the present invention is directed to a nucleic acid molecule comprising a sequence of nucleotides substantially as set forth in SEQ ID NO: 3 or a nucleotide sequence having at least 50% similarity thereto or a nucleotide sequence capable of hybridizing to SEQ ID NO:3 or a complementary form thereof under low stringency conditions wherein said nucleic acid molecule corresponds to a uterine estrogen agonist-inducible genetic sequence.
  • Alternative percentage similarities contemplated and encompassed by the present invention include at least about 60%, at least about 70%, at least about 80%, at least about 90% and at least about 95% or above such as 96%, 97%, 98% or 99%.
  • the percentage similarity is measured with respect to the entire nucleotide sequence of SEQ ID NO:l or SEQ ID NO:3.
  • the percentage similarity may also be relative to at least about 30 contiguous nucleotides of SEQ ID NO:l or SEQ ID NO:3 and preferably at least 30 contiguous nucleotides within a region characteristic of said uterine estrogen agonist-inducible genetic sequence.
  • An example of the latter is a region encoding a CUB domain, a hydrophobic transmembrane domain, an anchor domain and/or a zona pellucida domain.
  • characteristic is not to imply that the sequence is unique to the uterine estrogen agonist-inducible genetic sequence, although such an aspect is encompassed by the present invention.
  • the “characteristic” may need only be present in the genetic sequence.
  • similarity includes exact identity between compared sequences at the nucleotide or amino acid level. Where there is non-identity at the nucleotide level, “similarity” includes differences between sequences which result in different amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels. Where there is non-identity at the amino acid level, “similarity” includes amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels, hi a particularly preferred embodiment, nucleotide and sequence comparisons are made at the level of identity rather than similarity.
  • references to describe sequence relationships between two or more polynucleotides or polypeptides include “reference sequence”, “comparison window”, “sequence similarity”, “sequence identity”, “percentage of sequence similarity”, “percentage of sequence identity”, “substantially similar” and “substantial identity”.
  • a “reference sequence” is at least 12 but frequently 15 to 18 and often at least 25 or above, such as 30 monomer units, inclusive of nucleotides and amino acid residues, in length. Because two polynucleotides may each comprise (1) a sequence (i.e.
  • sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity.
  • a “comparison window” refers to a conceptual segment of typically 12 contiguous residues that is compared to a reference sequence.
  • the comparison window may comprise additions or deletions (i.e. gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • Optimal alignment of sequences for aligning a comparison window may be conducted by computerised implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and the best alignment (i.e., resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected.
  • GAP Garnier et al.
  • sequence similarity and “sequence identity” as used herein refers to the extent that sequences are identical or functionally or structurally similar on a nucleotide-by- nucleotide basis or an amino acid-by-amino acid basis over a window of comparison.
  • a “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g. A, T, C, G, I) or the identical amino acid residue (e.g.
  • sequence identity will be understood to mean the "match percentage” calculated by the D ⁇ ASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA) using standard defaults as used in the reference manual accompanying the software. Similar comments apply in relation to sequence similarity.
  • Reference herein to a low stringency includes and encompasses from at least about 0 to at least about 15% v/v formamide and from at least about 1 M to at least about 2 M salt for hybridization, and at least about 1 M to at least about 2 M salt for washing conditions.
  • low stringency is at from about 25-30°C to about 42°C. The temperature may be altered and higher temperatures used to replace formamide and/or to give alternative stringency conditions.
  • Alternative stringency conditions may be applied where necessary, such as medium stringency, which includes and encompasses from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5 M to at least about 0.9 M salt for hybridization, and at least about 0.5 M to at least about 0.9 M salt for washing conditions, or high stringency, which includes and encompasses from at least about 31% v/v to at least about 50% v/v formamide and from at least about 0.01 M to at least about 0.15 M salt for hybridization, and at least about 0.01M to at least about 0.15M salt for washing conditions.
  • medium stringency which includes and encompasses from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5 M to at least about 0.9 M salt for hybridization, and at least about 0.5 M to at least about 0.9 M salt for washing conditions
  • high stringency which includes and encompasses from at least about 31% v/v to at least about 50% v/v form
  • T m of a duplex D ⁇ A decreases by 1°C with every increase of 1% in the number of mismatch base pairs (60).
  • Formamide is optional in these hybridization conditions.
  • particularly preferred levels of stringency are defined as follows: low stringency is 6 x SSC buffer, 0.1% w/v SDS at 25-42°C; a moderate stringency is 2 x SSC buffer, 0.1% w/v SDS at a temperature in the range 20°C to 65°C; high stringency is 0.1 x SSC buffer, 0.1% w/v SDS at a temperature of at least 651°C.
  • the nucleic acid molecule of the present invention and in particular UO-44 is generally in isolated form. This means that it has undergone at least one purification or isolation step from a biological sample.
  • the nucleic acid molecule of the present invention may, therefore, be referred to as an "isolated nucleic acid molecule", a “biologically pure nucleic acid molecule” and/or an “isolated, purified nucleic acid molecule” amongst other suitable terms.
  • An isolation step includes centrifugation, precipitation, chromatographic separation as well as electrophoretic separation.
  • the subject nucleic acid molecule and in particular UO-44 may correspond to the genomic form of the uterine estrogen agonist-inducible genetic sequence or it may comprise one or more nucleotide substitutions, additions and/or deletions. These may be naturally occurring alterations in the nucleotide sequence or they may be artificially induced by any number of techniques.
  • the nucleic acid molecule may comprise a fusion of heterologous sequences such as from different homologs from different individuals or different mammalian sources.
  • One particularly useful form of the nucleic acid molecule is a "humanized" form of a non-human derived nucleic acid molecule. Conveniently, all such nucleotide modifications are encompassed by nucleic acid molecules being capable of hybridizing to SEQ ID NO:l or SEQ ID NO:3 or its complementary form under low stringency conditions.
  • the nucleic acid molecule of the present invention may be in single stranded or double stranded form and may be alone or in combination with a vector.
  • another aspect of the present invention relates to a genetic construct comprising a nucleotide sequence corresponding to a uterine estrogen agonist-inducible genetic sequence.
  • the nucleotide sequence is operably linked to a promoter sequence.
  • the promoter sequence may be naturally associated with the uterine estrogen agonist-inducible sequence or it may be another promoter.
  • Other nucleotide sequences may be located on the genetic construct such as those encoding a reporter molecule or selectable molecule and/or those involved in regulation of the subject nucleotide sequence.
  • the genetic construct comprises UO-44 or a derivative or homologue thereof.
  • a “derivative” includes a mutant, part, portion or fragment of UO-44.
  • nucleic acid molecule of the present invention and particularly UO-44 or its derivatives or homologs is conveniently used to produce a recombinant polypeptide, i.e. a uterine estrogen agonist-mediating polypeptide.
  • another aspect of the present invention contemplates a method for producing a uterine estrogen agonist-mediating polypeptide said method comprising introducing into a cell a nucleic acid molecule comprising a nucleotide sequence corresponding to a uterine estrogen agonist-inducible genetic sequence and subjecting said cell to conditions sufficient to permit expression of the nucleotide sequence and then recovering the polypeptide or membrane portions comprising same.
  • the cell may be a prokaryotic or eukaryotic cell. Examples of the latter include insect cells, mammalian cells, uterine cell lines and yeast cells.
  • the nucleotide sequence is UO-44 or its derivatives or homologs. Accordingly, another aspect of the present invention provides a polypeptide comprising a sequence of amino acids corresponding to a uterine estrogen agonist-mediating polypeptide.
  • polypeptide includes a peptide and protein.
  • the uterine estrogen agonist-mediating polypeptide is present in estrogen agonist treated ovariectomized uterus cells or tissue but substantially not or in low amounts in non-estrogen agonist treated ovariectomized uterine tissue.
  • the polypeptide is encoded by UO-44 or its derivative or homologue.
  • another aspect of the present invention is directed to a polypeptide comprising sequence of amino acids encoded by the nucleotide sequence substantially as set forth in SEQ ID NO:l or a nucleotide sequence having at least 50% similarity thereto or a nucleotide sequence capable of hybridizing SEQ ID NO:l under low stringency conditions.
  • the present invention provides a polypeptide comprising a sequence of amino acids substantially as set forth in SEQ ID NO:2 or an amino acid sequence having at least about 50% similarity thereto.
  • the present invention provides a polypeptide comprising a sequence of amino acids substantially as set forth in SEQ ID NO:2 or an amino acid sequence having at least about 50% similarity thereto and which polypeptide corresponds to a uterine estrogen agonist-mediating polypeptide or a mutant, derivative, homologue or analogue of said polypeptide.
  • the present invention is directed to a polypeptide comprising sequence of amino acids encoded by the nucleotide sequence substantially as set forth in
  • SEQ ID NO: 3 or a nucleotide sequence having at least 50% similarity thereto or a nucleotide sequence capable of hybridizing SEQ ID NO: 3 under low stringency conditions.
  • the present invention provides a polypeptide comprising a sequence of amino acids substantially as set forth in SEQ ID NO:4 or an amino acid sequence having at least about 50% similarity thereto.
  • the present invention provides a polypeptide comprising a sequence of amino acids substantially as set forth in SEQ ID NO:4 or an amino acid sequence having at least about 50% similarity thereto and which polypeptide corresponds to a uterine estrogen agonist-mediating polypeptide or a mutant, derivative, homologue or analogue of said polypeptide.
  • the present invention encompasses a range of derivatives of the uterine estrogen agonist- mediating polypeptide. These are all encompassed by the term "UO-44" or reference to the UO-44 polypeptide.
  • Derivatives include fragments, parts, portions, mutants, homologs and analogs of the subject polypeptide. Derivatives also include single or multiple amino acid substitutions, deletions and/or additions to the subject polypeptide. "Additions" to amino acid sequences or nucleotide sequences include fusions with other peptides, polypeptides or proteins or fusions to nucleotide sequences.
  • Reference herein to the "uterine estrogen agonist-mediating polypeptide" or reference to the "subject polypeptide” includes reference to all derivatives thereof including functional and non-functional derivatives.
  • Analogs of the subject polypeptide contemplated herein include, but are not limited to, modification to side chains, incorporating of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the proteinaceous molecule or their analogs.
  • side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH 4 ; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH .
  • amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH 4 ; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation
  • the guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.
  • the carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitization, for example, to a corresponding amide.
  • Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4- chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid, phenylmercury chloride, 2- chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.
  • Tryptophan residues may be modified by, for example, oxidation with N- bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides.
  • Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.
  • Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate.
  • Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3- hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids.
  • a list of unnatural amino acid, contemplated herein is shown in Table 1.
  • peptides can be conformationally constrained by, for example, incorporation of C ⁇ and N c rmethylamino acids, introduction of double bonds between C a and C ⁇ atoms of amino acids and the formation of cyclic peptides or analogs by introducing covalent bonds such as forming an amide bond between the N and C termini, between two side chains or between a side chain and the N or C terminus.
  • the present invention further contemplates chemical analogs of the subject polypeptide capable of acting as antagonists or agonists of the polypeptide or which can act as functional analogs of polypeptide.
  • Chemical analogs may not necessarily be derived from the instant polypeptide but may share certain conformational similarities. Alternatively, chemical analogs may be specifically designed to mimic certain physiochemical properties of the subject polypeptide. Chemical analogs may be chemically synthesised or may be detected following, for example, natural product screening. The latter refers to molecules identified from various environmental sources such as river beds, coral, plants, microorganisms and insects.
  • modifications may be important to stabilize the subject polypeptide if administered to an individual or for use as a diagnostic reagent.
  • Other derivatives contemplated by the present invention include a range of glycosylation variants from a completely unglycosylated molecule to a modified glycosylated molecule. Altered glycosylation patterns may result from expression of recombinant molecules in different host cells.
  • Non-conventional Code Non-conventional Code amino acid amino acid
  • D-N-methyltryptophan Dnmtrp N-(l-methylethyl)glycine Nval
  • D-N-methyltyrosine Dnmtyr N-methyla-napthylalanine Nmanap
  • the identification of the subject nucleic acid molecule and its corresponding polypeptide permits the generation of a range of therapeutic molecules capable of modulating estrogen agonist-mediating cell growth and proliferation in the uterus including ovaries.
  • Modulators contemplated by the present invention includes agonists and antagonists of gene expression or polypeptide activity.
  • Antagonists include antisense molecules, ribozymes, co- suppression molecules, antibodies inhibitor peptide fragments and soluble receptors.
  • Agonists include molecules which increase promoter activity or interfere with negative regulatory mechanisms.
  • Another embodiment of the present invention contemplates a method for modulating expression of a uterine estrogen agonist-inducible genetic sequence in a mammal such as a human, primate or laboratory test animal, said method comprising contacting a gene corresponding to said genetic sequence with an effective amount of a modulator of gene expression for a time and under conditions sufficient to up-regulate or down-regulate or otherwise modulate expression of the uterine estrogen agonist inducible genetic sequence.
  • Another aspect of the present invention contemplates a method of modulating activity of the uterus estrogen agonist-mediating polypeptide in a mammal such as a human, primate or laboratory test animal, said method comprising administering to said mammal a modulating effective amount of a molecule for a time and under conditions sufficient to increase or decrease activity of the uterine estrogen agonist-inducible genetic sequence.
  • the molecule may be a proteinaceous molecule or a chemical entity and may also be a derivative of the subject polypeptide or its ligand or a chemical analogue or truncation mutant of said polypeptide or its ligand. Modulating levels of the instant polypeptide or its genetic sequence may be important in modulating uterine growth and development as well as in the treatment of uterine and ovarian cancers.
  • the present invention contemplates a composition comprising a uterine estrogen agonist-mediating polypeptide and one or more pharmaceutically acceptable carriers and/or diluents. These components are referred to as the "active ingredients”.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) and sterile powders for the extemporaneous preparation of sterile injectable solutions. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as licithin.
  • microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by sterilization.
  • the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient.
  • the active ingredients When the active ingredients are suitably protected they may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets.
  • the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • Such compositions and preparations should contain at least 1% by weight of active compound.
  • the percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit.
  • the amount of active compound in such therapeutically useful compositions in such that a suitable dosage will be obtained.
  • Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 0.01 ⁇ g and about 2000 mg of active compound per subject.
  • Alternative amounts include between about 1.0 peg and about 1500 ng, between about 1 ⁇ g and about 1000 mg and between about 10 ⁇ g and about 500 mg per subject.
  • Equivalent doses may alsobe expressed per kilogram of body weight.
  • the tablets, troches, pills, capsules and the like may also contain the components as listed hereafter: a binder such as gum, acacia, corn starch or gelatin; xcipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such a sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring.
  • a binder such as gum, acacia, corn starch or gelatin
  • xcipients such as dicalcium phosphate
  • a disintegrating agent such as corn starch, potato starch, alginic acid and the like
  • a lubricant such as magnesium stearate
  • a sweetening agent such as sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil
  • tablets, pills, or capsules may be coated with shellac, sugar or both.
  • a syrup or elixir may contain the active compound, sucrose as a sweetemng agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour.
  • any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed.
  • the active compound(s) may be incorporated into sustained-release preparations and formulations.
  • Pharmaceutically acceptable carriers and/or diluents include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
  • the principal active ingredient is compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in dosage unit form.
  • a unit dosage form can, for example, contain the principal active compound in amounts ranging from 0.01 ⁇ g to about 2000 mg. Expressed in proportions, the active compound is generally present in from about 0.5 ⁇ g to about 2000 mg/ml of carrier.
  • the dosages are determined by reference to the usual dose and manner of administration of the said ingredients.
  • amounts administered may be represented in terms of amounts/kg body weight, hi this case, amounts range from about 0.001 ⁇ g to about 1000 mg/kg body weight may be administered 500 mg/kg body weight or about 10.01 ⁇ g to about or above 0.1 ⁇ g to about 250 mg/kg body weight are contemplated by the present invention.
  • the pharmaceutical composition may also comprise genetic molecules such as a vector capable of transfecting target cells where the vector carries a nucleic acid molecule capable of modulating expression of the uterus estrogen agonist-inducible genetic sequence.
  • the vector may, for example, be a viral vector.
  • Still another aspect of the present invention is directed to antibodies to the uterine estsrogen agonist-mediating polypeptide and its derivatives.
  • Such antibodies may be monoclonal or polyclonal and may be selected from naturally occurring antibodies to the polypeptide or may be specifically raised to the polypeptide or its derivatives thereof.
  • the antibodies and/or recombinant polypeptide or its derivatives of the present invention are particularly useful as therapeutic or diagnostic agents.
  • a recombinant polypeptide and its derivatives can be used to screen for naturally occurring antibodies to estrogen agonist-mediating polypeptides. These may occur, for example in some autoimmune diseases.
  • specific antibodies can be used to screen for the presence of levels of estrogen agonist-mediating polypeptides.
  • Techniques for such assays are well known in the art and include, for example, sandwich assays and ELISA. Knowledge of levels of subject polypeptides may be important for diagnosis of certain cancers or a predisposition to cancers or for monitoring certain therapeutic protocols.
  • Antibodies to the subject polypeptides of the present invention may be monoclonal or polyclonal. Alternatively, fragments of antibodies may be used such as Fab fragments. Furthermore, the present invention extends to recombinant and synthetic antibodies and to antibody hybrids. A "synthetic antibody” is considered herein to include fragments and hybrids of antibodies.
  • the antibodies of this aspect of the present invention are particularly useful for immunotherapy and may also be used as a diagnostic tool for assessing apoptosis, cancer, tissue regeneration or development, muscle development or health of neural tissue or for monitoring the program of a therapeutic mitin.
  • specific antibodies can be used to screen for the subject polypeptides.
  • the latter would be important, for example, as a means for screening for levels of a polypeptide in a cell extract or other biological fluid or purifying a polypeptide made by recombinant means from culture supernatant fluid.
  • Techniques for the assays contemplated herein are known in the art and include, for example, sandwich assays and ELISA.
  • second antibodies (monoclonal, polyclonal or fragments of antibodies or synthetic antibodies) directed to the first mentioned antibodies discussed above. Both the first and second antibodies may be used in detection assays or a first antibody may be used with a commercially available anti- immunoglobulin antibody.
  • An antibody as contemplated herein includes any antibody specific to any region of the polypeptide.
  • Both polyclonal and monoclonal antibodies are obtainable by immunization with the enzyme or protein and either type is utihzable for immunoassays.
  • the methods of obtaining both types of sera are well known in the art.
  • Polyclonal sera are less preferred but are relatively easily prepared by injection of a suitable laboratory animal with an effective amount of subject polypeptide, or antigenic parts thereof, collecting serum from the animal, and isolating specific sera by any of the known immunoadsorbent techniques.
  • antibodies produced by this method are utihzable in virtually any type of immunoassay, they are generally less favoured because of the potential heterogeneity of the product.
  • the use of monoclonal antibodies in an immunoassay is particularly preferred because of the ability to produce them in large quantities and the homogeneity of the product.
  • the preparation of hybridoma cell lines for monoclonal antibody production derived by fusing an immortal cell line and lymphocytes sensitized against the immunogenic preparation can be done by techniques which are well known to those who are skilled in the art.
  • Another aspect of the present invention contemplates a method for detecting a uterus estrogen agonist-mediating polypeptide in a biological sample from a subject said method comprising contacting said biological sample with an antibody specific for said uterine estrogen agonist-inducible genetic sequence or its derivatives or homologs for a time and under conditions sufficient for an antibody-polypeptide complex to form, and then detecting said complex.
  • the presence of the instant polypeptide may be accomplished in a number of ways such as by Western blotting and ELISA procedures.
  • a wide range of immunoassay techniques are available as can be seen by reference to U.S. Patent Nos. 4,016,043, 4, 424,279 and 4,018,653.
  • Sandwich assays are among the most useful and commonly used assays and are favoured for use in the present invention.
  • a second antibody specific to the antigen, labelled with a reporter molecule capable of producing a detectable signal is then added and incubated, allowing time sufficient for the formation of another complex of antibody-antigen-labelled antibody. Any unreacted material is washed away, and the presence of the antigen is determined by observation of a signal produced by the reporter molecule. The results may either be qualitative, by simple observation of the visible signal, or may be quantitated by comparing with a control sample containing known amounts of hapten. Variations on the forward assay include a simultaneous assay, in which both sample and labelled antibody are added simultaneously to the bound antibody.
  • the sample is one which might contain a subject polypeptide including membrane preparation or tissue biopsy.
  • the sample is, therefore, generally a biological sample comprising biological fluid but also extends to preparations containing membranes.
  • a first antibody having specificity for the instant polypeptide or antigenic parts thereof is either covalently or passively bound to a solid surface.
  • the solid surface is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
  • the solid supports may be in the form of tubes, beads, discs of microplates, or any other surface suitable for conducting an immunoassay.
  • the binding processes are well-known in the art and generally consist of cross-linking covalently binding or physically adsorbing, the polymer-antibody complex is washed in preparation for the test sample.
  • an aliquot of the sample to be tested is then added to the solid phase complex and incubated for a period of time sufficient (e.g. 2-40 minutes or where more convenient, overnight) and under suitable conditions (e.g. for about 20°C to about 40°C) to allow binding of any subunit present in the antibody.
  • the antibody subunit solid phase is washed and dried and incubated with a second antibody specific for a portion of the hapten.
  • the second antibody is linked to a reporter molecule which is used to indicate the binding of the second antibody to the hapten.
  • An alternative method involves immobilizing the target molecules in the biological sample and then exposing the immobilized target to specific antibody which may or may not be labelled with a reporter molecule. Depending on the amount of target and the strength of the reporter molecule signal, a bound target may be detectable by direct labelling with the antibody.
  • a second labelled antibody specific to the first antibody is exposed to the target-first antibody complex to form a target-first antibody-second antibody tertiary complex. The complex is detected by the signal emitted by the reporter molecule.
  • reporter molecule as used in the present specification, is meant a molecule which, by its chemical nature, provides an analytically identifiable signal which allows the detection of antigen-bound antibody. Detection may be either qualitative or quantitative.
  • the most commonly used reporter molecules in this type of assay are either enzymes, fluorophores or radionuclide containing molecules (i.e. radioisotopes) and chemiluminescent molecules, hi the case of an enzyme immunoassay, an enzyme is conjugated to the second antibody, generally by means of glutaraldehyde or periodate.
  • glutaraldehyde or periodate As will be readily recognized, however, a wide variety of different conjugation techniques exist, which are readily available to the skilled artisan.
  • Commonly used enzymes include horseradish peroxidase, glucose oxidase, beta-galactosidase and alkaline phosphatase, amongst others.
  • the substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable colour change. Examples of suitable enzymes include alkaline phosphatase and peroxidase. It is also possible to employ fluorogenic substrates, which yield a fluorescent product rather than the chromogenic substrates noted above. In all cases, the enzyme-labelled antibody is added to the first antibody hapten complex, allowed to bind, and then the excess reagent is washed away. A solution containing the appropriate substrate is then added to the complex of antibody-antigen- antibody.
  • the substrate will react with the enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantitated, usually spectrophotometrically, to give an indication of the amount of hapten which was present in the sample.
  • Reporter molecule also extends to use of cell agglutination or inhibition of agglutination such as red blood cells on latex beads, and the like.
  • fluorescent compounds such as fluorescein and rhodamine
  • fluorescein and rhodamine may be chemically coupled to antibodies without altering their binding capacity.
  • the fluorochrome-labelled antibody When activated by illumination with light of a particular wavelength, the fluorochrome-labelled antibody adsorbs the light energy, inducing a state to excitability in the molecule, followed by emission of the light at a characteristic colour visually detectable with a light microscope.
  • the fluorescent labelled antibody is allowed to bind to the first antibody- hapten complex. After washing off the unbound reagent, the remaining tertiary complex is then exposed to the light of the appropriate wavelength the fluorescence observed indicates the presence of the hapten of interest.
  • Immunofluorescene and EIA techniques are both very well established in the art and are particularly preferred for the present method. However, other reporter molecules, such as radioisotope, chemiluminescent or bioluminescent molecules, may also be employed.
  • the present invention also contemplates genetic assays such as involving PCR analysis to detect a uterus estrogen agonist-inducible genetic sequence its derivatives.
  • Alternative methods or methods which may be used in conjunction include direct nucleotide sequencing or mutation scanning such as single stranded conformation polymorphoms analysis (SSCP) as well as specific oligonucleotide hybridization.
  • SSCP single stranded conformation polymorphoms analysis
  • UO-44 permits the monitoring of uterine growth and development and also provides a marker for uterine and ovarian cancers.
  • the UO-44 has been cloned from rat and, in particular, Rattus norvegirus (i.e. rUO-44) as well as from humans (i.e. hUO-44). This is done, however, with the understanding that the present invention extends to UO-44 homologs from any species but in particular from primate and livestock animals.
  • the present invention extends to a UO-44 homologue from a primate or livestock animal, said homologue comprising a nucleotide sequence capable of hybridizing to SEQ ID NO:l and/or SEQ ID NO: 3 or their complementary forms under stringency conditions.
  • the present invention further extends to promoter and other regulatory regions associated with the UO-44 gene as well as genomic clones. Genomic clones are particularly useful in gene therapy.
  • the present invention further extends to genetically modified animals such as mice, rats, rabbits, sheep or non-human primates which are unable to express UO-44 or which have modified levels of expression of UO-44 genetic material. Such animals and in particular mice and rats are excellent models for screening, inter alia, for antagonists and agonists.
  • ICI 182780 (AstraZeneca Pharmaceuticals) was supplied at a concentration of 50 mg/ml in castor oil solution.
  • Ovariectomized rats were either untreated (OVX) or treated with 5 mgkg BW tamoxifen per day (OVX-TAM) for 14 days.
  • Total RNA was isolated from uteri using RNAzol prernix solution and RNAzol B method (Tel-Test, Friendswood, TX) as previously described (49). Differential display was performed using RNA derived from uteri of OVX and OVX-TAM rats according to the protocol supplied with the RNAmap (trademark) kit (GenHunter Corp., Nashville, TN).
  • RNA reverse transcribed with 300 units of Moloney murine leukemia virus reverse transcriptase (Pharmacia) in the presence of 1 niM T 12 MG, T 12 MA, T 12 MT, or T 12 MC primer (GenHunter) where M is a mixture containing dG, dA, and dC.
  • Double stranded cD ⁇ A was size enriched and transformed into TOPI OF' cells after ligation into the vector.
  • the 320 bp D ⁇ A probe of rat UO-44 cD ⁇ A was used to screen this rat uterine cD ⁇ A library as described (50). Clones identified by this probe were isolated and sequenced by the Sanger dideoxy chain termination method and their nucleotide sequences were compared with those deposited in the GenBank and EMBL databases.
  • recombinant plasmid pcD ⁇ A3.0 containing a 500-bp UO-44 fragment was linearized to generate sense and antisense digoxigenin-labeled RNA probes using Dig RNA Labeling kit (Boehringer Mannheim). Serial 7-8 ⁇ m OCT-frozen sections from either uterus or ovary were heated for 2 min at 50°C and dried for 30 min. Prehybridization, hybridization, posthybridization and immunological detection were performed according to the manufacturer's protocol and these sections were subsequently counterstained with haematoxylin. EXAMPLE 5 Northern blotting
  • UO-44 cDNA The entire coding region of UO-44 cDNA was cloned into the mammalian expression vector pcDNA3.1 His (Invitrogen, Carlsbad, CA) to create the UO-44-pcDNA3.1/His expression vector.
  • the UO-44-pcDNA3.1/His sequence was confirmed by sequencing.
  • MCF-7 cells were seeded at a density of 2 x 10 5 in 100 mm culture dishes in 90% ⁇ -MEM (Life Technologies, Inc.) containing 10% w/v FCS with Garamycin 24 hr prior to transfection.
  • Cells were transfected with 5 ⁇ g of UO-44-pDNA3.1/His DNA or pDNA3.1/His control plasmid DNA and 28 ⁇ l of Lipofectamine reagent (Life Technologies) following recommendations of the manufacturer. Forty-eight hours following transfection, cells were subcultured at 1:10 and replaced with growth medium containing 800 ⁇ g/ml G418 (Calbiochem, La Jolla, CA). After 4 weeks, clones were isolated, expanded and assayed for UO-44 expression by Western blot analysis.
  • Plasma membrane-enriched subcellular fractions and cytosol were prepared by differential centrifugation as described previously (52). Plasma membrane and cytosolic proteins were subjected to Western blot analysis as described (53). Blots were incubated with mouse anti 6-Histidine antibody (Epitope Tagging) Ab-1 (NeoMarkers, Union City, CA) (1:500 dilution) and horseradish peroxidase-conjugated donkey anti-mouse secondary antibody (1:7500). Blots were visualized with a chemiluminescent detection system (ECL, Amersham) and exposed to film for 10 sec to 45 sec.
  • ECL chemiluminescent detection system
  • differential display methodology Using differential display methodology, several differentially displayed bands representing cDNA corresponding to gene whose expression was up-regulated during tamoxifen treatment were isolated.
  • One of the bands which was present in the uteri of tamoxifen-treated rats but disappeared in uteri derived from ovariectomized rats, was selected for further characterization ( Figure 1 A).
  • This 320 bp DNA fragment was used to probe Northern blots of mRNA obtained from uteri of OVX and OVX-tamoxifen-treated rats.
  • the full-length UO-44 cDNA contains 2282 bp of nucleotide sequence.
  • An initiator ATG codon (position 253) is followed by a single open reading frame of 607 amino acids with a calculated molecular weight of 68639 Da.
  • the ATG initiation site is contained in the sequence for initiation by eukaryotic ribosomes described by Kozak (55).
  • the open reading frame ends in a TGA teiniinator codon at position 2074 followed by 208 nucleotides in the 3' untranslated region.
  • UO-44 Within the first 265 amino acids of UO-44, two regions were identified that bear homology to the CUB motifs (complement subcomponents Clr/Cls, Uegf protein and bone morphogenetic protein) (56). The first CUB domain began at Cys-32 and the second at Cys-154. UO-44 also contained a zona pellucida domain at amino acids 276-523. The UO-44 amino acid sequence predicted a membrane protein with two transmembrane helices. The hydrophobic transmembrane region was 13 amino acids in length and was located between amino acids 5 and 17, while the anchor transmembrane region was 19 amino acids in length and is located between amino acids 571 and 589. There was a putative transmembrane domain near the carboxyl terminus. UO-44 teiminated in a short 19 amino acid polypeptide presumably positioned within the cytoplasm.
  • RNA obtained from tissues of mature female rats was analyzed by Northern blotting.
  • Figure 2 shows that transcription of the UO-44 gene was observed only in uterus and ovary.
  • UO-44 mRNA levels in adipose tissue, mammary gland, liver, kidney, muscle, heart, stomach, small intestine, spleen, brain, pituitary, and muscle were undetectable, suggesting that the UO-44 gene may be expressed at a very low level or not at all in these tissues. Since this gene is highly expressed in the uterus and ovary, it has been designated Uterine-Ovarian specific gene 44 or UO-44.
  • ICI 182780 was a very potent inhibitor of UO-44 gene expression, completely abolishing UO-44 gene expression in the uterus at the dose of 1 mg per kg body weight per week as compared with the ovary-intact uterus ( Figure 5B). There is a positive correlation between UO-44 gene expression and uterine weight.
  • Uterine weight is known to increase by the influence of growth hormone (49), and this led the inventors to investigate whether UO-44 gene expression in the uterus was under the regulation of growth hormone.
  • Northern blot analysis was performed using uteri and ovaries obtained from hypophysectomized (Hypox) rats treated with vehicle or various doses of recombinant human growth hormone. Comparing to uterine weight of ovary intact rats ( Figure 5), hypophysectomy caused a sigmficant reduction in uterine weight which was coincident with the disappearance of UO-44 mRNA ( Figure 6). Upon administration of growth hormone, UO-44 gene expression in the uterus was restored.
  • Figure 5 showed that UO-44 mRNA was undetectable in uteri derived from ovariectomized rats and was expressed followed tamoxifen treatment.
  • in situ hybridization was performed on sections of uteri derived from OVX-tamoxifen-treated rats using an antisense RNA probe specific for rat UO-44. High levels of UO-44 were detected in the luminal epithelial cells and glandular population of the uteri following tamoxifen treatment ( Figures 8A and 8B).
  • MCF-7 cells were transfected with a mammalian expression vector containing full length UO- 44 cDNA (UO-44 ⁇ cDNA3.1/His) or control pcDNA3.1 His vector. As shown in Figure 10,
  • 6-Histidine antibody recognized a protein of approximately 68-69 kDa in plasma membrane- enriched subcellular fractions of UO-44 transfectants but not in the cytosol. No protein of identical size was detected in mock-transfected cells.
  • the murine UO-44 cDNA was used to identify a human homolog. Using the murine coding region as a probe in a screening procedure, a full length human UO-44 (hUO-44) cDNA from a human uterus library was identified, cloned and sequenced to its entirety.
  • the full length hUO-44 possess an open reading frame (ORF) of 357 amino acids with a calculated molecular weight of 40.17 kDa and a pi of 8.19.
  • ORF open reading frame
  • the nucleotide and corresponding amino acid sequence is set forth in SEQ ID NO:3 and SEQ ID NO:4, respectively.
  • the hUO-44 gene is also present in a human placenta library. Data indicate that the hUO- 44 gene is approximately 7-8 kb inclusive of the promoter regions. There are also six exons encoding the full length hUO-44 protein and these exons were also found to comply with the consensus exon-intron junctions.
  • BLAST data from the human genome sequence database from NCBI revealed that the hUO-44 gene is aligned to a specific contig clone from chromosome 10 with six BLAST hits, all with a homology of greater than 97% confidence. These six BLAST hits coincide well with the exons of hUO-44.
  • the contig clone where hUO-44 gene resides is known as NT008720.
  • a gene known a the DMBTl gene (deleted in malignant brian tumor gene 1).
  • DMBTl gene (deleted in malignant brian tumor gene 1).
  • tumor suppressor genes at chromosome lOq are involved in the carcinogenesis of brain tumors, melanomas and carcinomas of the lung, the prostate, the pancreas and the endometrium.
  • the DMBTl gene has at least 54 exons which span a genomic region of about 80 kb. This organisation of the domain structure of the DMBTl protein reveals certain degrees of similarity to that of the hUO-44 protein with the existence of the ZP and transmembrane domains. The presence of the CUB domains is also pronounced to that of the murine homolog which possess two CUB domains.
  • hUO-44 may also function as a tumor suppressor gene.

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Abstract

Molécule d'acide nucléique qui comporte une séquence nucléotidique correspondant à une séquence génétique induite par un agoniste d'oestrogène dans des cellules provenant de l'utérus. La molécule d'acide nucléique selon la présente invention code une protéine destinée à entraîner les hormones oestrogéniques et autres agonistes d'oestrogène à induire la prolifération épithéliale et la différenciation en tissus utérins et en tissus ovariens. La présente invention concerne donc un moyen de surveiller la croissance et le développement utérins et ovariens ainsi que le niveau d'activité oestrogénique dans des tissus, dont des tissus cancéreux, en mesurant par exemple l'expression de la molécule d'acide nucléique du sujet et/ou en détectant le taux du polypeptide induisant l'agoniste d'oestrogène codé par ladite molécule. La présente invention concerne encore des compositions contenant le polypeptide décrit ou ses dérivés, homologues, analogues ou agonistes. Elle concerne également une méthode de modulation de la croissance de tissus utérins et/ou ovariens dont des tissus cancéreux provenant d'utérus ou d'ovaires par administration d'un agoniste ou antagoniste de l'expression de la molécule d'acide nucléique du sujet ou de l'activité du polypeptide codé par ladite molécule. La molécule d'acide nucléique selon la présente invention fournit également directement ou indirectement via son produit d'expression un moyen permettant de diagnostiquer, de détecter ou de surveiller des cancers de l'utérus et/ou de l'ovaire. La présente invention concerne enfin un promoteur et/ou d'autres régions régulatrices associées à l'expression de la molécule d'acide nucléique du sujet.
PCT/AU2001/000379 2000-04-04 2001-04-04 Molecule d'acide nucleique codant une proteine uterine pouvant etre induite par un agoniste d'oestrogene WO2001075099A1 (fr)

Priority Applications (1)

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AU43962/01A AU4396201A (en) 2000-04-04 2001-04-04 Nucleic acid molecule encoding a uterine estrogen agonist- inducible protein

Applications Claiming Priority (4)

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US19456600P 2000-04-04 2000-04-04
US60/194,566 2000-04-04
AUPQ9471 2000-08-15
AUPQ9471A AUPQ947100A0 (en) 2000-08-15 2000-08-15 A nucleic acid molecule and uses therefor

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Publication number Priority date Publication date Assignee Title
US20150072349A1 (en) * 2012-03-16 2015-03-12 University Health Network Cancer Biomarkers and Methods of Use

Citations (1)

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WO1999014328A2 (fr) * 1997-09-17 1999-03-25 Genentech, Inc. Polypeptides secretes et transmembranaires et acides nucleiques les codant

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WO1999014328A2 (fr) * 1997-09-17 1999-03-25 Genentech, Inc. Polypeptides secretes et transmembranaires et acides nucleiques les codant

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Title
CHEN D. ET AL.: "Cloning and uterus/oviduct-specific expression of a novel estrogen-regulated gene (ERG1)", J. BIOL. CHEM., vol. 274, no. 45, 1999, pages 32215 - 32224 *
DATABASE PROTEIN [online] 12 October 2000 (2000-10-12), NG Y.C. ET AL.: "Uterus-ovary specific putative transmembrane protein UO (homo sapiens)", XP002946722, Database accession no. AAG23215 *
DATABASE PROTEIN [online] 2 October 1997 (1997-10-02), HUYNH T.H. ET AL.: "Uterus-ovary specific putative transmembrane protein (rattus norvegicus)", XP002946723, Database accession no. AAB1895 *
KASIK J.W.: "a cDNA cloned from pregnant mouse uterus exhibits temporo-spatial expression and predicts a novel protein", BIOCHEM. J., vol. 380, 1998, pages 947 - 950 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150072349A1 (en) * 2012-03-16 2015-03-12 University Health Network Cancer Biomarkers and Methods of Use

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