WO2000078955A9 - Inhibiteurs de croissance des cellules epitheliales - Google Patents

Inhibiteurs de croissance des cellules epitheliales

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Publication number
WO2000078955A9
WO2000078955A9 PCT/US2000/016900 US0016900W WO0078955A9 WO 2000078955 A9 WO2000078955 A9 WO 2000078955A9 US 0016900 W US0016900 W US 0016900W WO 0078955 A9 WO0078955 A9 WO 0078955A9
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WO
WIPO (PCT)
Prior art keywords
prostate
pmamm
cells
mammastatin
ecgi
Prior art date
Application number
PCT/US2000/016900
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English (en)
Other versions
WO2000078955A1 (fr
Inventor
Paul R Ervin Jr
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Biotherapies Inc
Paul R Ervin Jr
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Filing date
Publication date
Priority to AU60523/00A priority Critical patent/AU783373B2/en
Application filed by Biotherapies Inc, Paul R Ervin Jr filed Critical Biotherapies Inc
Priority to KR1020017016294A priority patent/KR20020016837A/ko
Priority to EP00946824A priority patent/EP1190054A1/fr
Priority to IL14714800A priority patent/IL147148A0/xx
Priority to BR0012301-3A priority patent/BR0012301A/pt
Priority to MXPA01013242A priority patent/MXPA01013242A/es
Priority to JP2001505697A priority patent/JP2003513615A/ja
Priority to CA002375498A priority patent/CA2375498A1/fr
Priority to NZ516545A priority patent/NZ516545A/en
Publication of WO2000078955A1 publication Critical patent/WO2000078955A1/fr
Priority to US10/028,952 priority patent/US6939714B2/en
Publication of WO2000078955A9 publication Critical patent/WO2000078955A9/fr
Priority to US11/058,923 priority patent/US20050169936A1/en

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to a family of epithelial cell growth inhibitors useful in the diagnosis and treatment of epithelial cell cancers.
  • Epithelial cell cancers for example, prostate cancer, breast cancer, colon cancer, lung cancer, pancreatic cancer, ovarian cancer, cancer of the spleen, testicular cancer, cancer of the thymus, etc.
  • diseases characterized by abnormal, accelerated growth of epithelial cells This accelerated growth initially causes a tumor to form. Eventually, metastasis to different organ sites can also occur.
  • Mammastatin A novel, specific, mammary cell growth inhibitor, Mammastatin, has recently been identified and characterized. Mammastatin has been expressed from variant clones, MammA (PCT7US97/18026, ATCC# 97451, deposited 22 February 1996); MammB (PCT/US97/27147, ATCC# PTA-2091, deposited 15 June 2000); and MammC, described in copending PCT application No. PCT/US00/16900 filed on even date herewith (ATCC# PTA-2090, deposited 15 June 2000).
  • MammA PCT7US97/18026, ATCC# 97451, deposited 22 February 1996
  • MammB PCT/US97/27147, ATCC# PTA-2091, deposited 15 June 2000
  • MammC described in copending PCT application No. PCT/US00/16900 filed on even date herewith (ATCC# PTA-2090, deposited 15 June 2000).
  • Mammastatin is produced and secreted by normal mammary cells, and is detected in blood samples of normal individuals. Blood concentrations of the mammary cell growth inhibitor, and particularly of the active, phosphorylated form of Mammastatin, are reduced or absent in breast cancer patients. Administration of protein comprising active Mammastatin (secreted from normal human breast cancer cells) is effective to reduce tumor size and number, and to prevent tumor growth in late stage cancer patients.
  • Epithelial cell growth inhibitors having similarity to Mammastatin have now been discovered, isolated, and characterized. These inliibitors bear partial sequence identity to Mammastatin at the 5' end of the sequence, and have little or no identity at the 3' end of the molecule.
  • the newly discovered family of epithelial cell growth inhibitors are differentially expressed in normal epithelial cell tissues, but not in cancerous epithelial cell tissues. Also, like
  • Mammastatin the newly discovered family of epithelial cell growth inhibitors are detected in blood samples taken from normal individuals, but not in the blood of patients with epithelial cell cancers, as shown in the Examples below.
  • ECGI epithelial cell growth inliibitors
  • the ECGI of the invention are expressed in normal epithelial cells but not in cancerous epithelial cells.
  • the Mammastatin-like ECGI proteins are encoded by nucleic acid sequences that hybridize to nucleic acid sequences encoding Mammastatin.
  • the ECGI proteins also bind anti-Mammastatin antibody.
  • a nucleic acid sequence encoding ECGI in prostate cells (PRT-6, SEQ ID NO: 4) has been isolated and characterized (PRT-6, ATCC# PTA-2092, deposited 15 June 2000), as described in the Examples below.
  • the ECGI of the invention are differently expressed by normal epithelial cells and not by cancerous epithelial cells, the presence or amount of the ECGI can be analyzed to diagnose cancer and/or to monitor treatment.
  • the inventive ECGI proteins and nucleic acids encoding them also provide useful therapeutic agents to inhibit epithelial cell growth, prevent tumor formation, and treat cancer.
  • Figure IA is a schematic diagram of an mRNA test panel showing locations of specific tissue mRNAs for analysis.
  • Figure IB is a computer scanned image of a Northern blot showing hybridization of Mammastatin nucleic acid sequence to niRNA from a variety of tissues according to the plan shown in Figure 1 A.
  • Figure 2 is a computer scanned image of a dot blot assay showing control, Mammastatin standard protein, serum samples from breast cancer patients, and conditioned medium from normal and cancerous human prostate cells probed with anti-Mammastatin antibody, 7G6.
  • Figure 3 is a computer scanned image of a Western blot assay, showing normal human mammary cell lysate (A), human prostate cancer LnCap cell lysate (B), MCF7 breast cancer cell lysate (C), and normal human prostate cell lysate (D) probed with anti-Mammastatin antibody, 7G6.
  • Figure 4 is a computer scanned image of a Western blot assay, showing cell lysates from normal prostate cells (A), LnCap prostate cancer cells (B), normal colon cells (C), and colon cancer cells (D) probed with anti-Mammastatin antibody, 7G6.
  • Figure 5 is a computer scanned image of a Western blot assay, showing cell lysates from human ovarian cancer cells (B), normal human ovarian cells (C), and normal human mammary cells (D) probed with anti-Mammastatin antibody, 7G6. Lane A contained molecular weight standards.
  • Figure 6 is a computer scanned image of a dot blot assay showing serum samples from healthy male adults (A,C,D) and from a prostate cancer patient (B) probed with anti-Mammastatin antibody, 7G6.
  • Figure 7 is a computer scanned image of a DNA gel containing putative prostate ECGF DNA clones.
  • Figure 8 is a diagramatic representation of Prostate ECGI and its structural relationship to other sequences.
  • Epithelial cell growth inhibitor (ECGI) proteins of the invention are defined herein to mean Mammastatin-like proteins produced by and active to inhibit the growth of normal epithelial cells. Active, inhibitory ECGI proteins of the invention are reduced or absent in cancerous epithelial cells.
  • the ECGI protein family disclosed herein appears to include inhibitors that are specific to each epithelial tissue, with little or no inhibitory activity across tissue types. As discussed more fully below, it is postulated that each ECGI protein contains a growth inhibitory domain and a tissue-specificity domain.
  • the ECGI proteins of the invention exhibit significant homology to Mammastatin, a mammary cell growth inhibitor produced by normal human mammary cells, and previously demonstrated be useful in the diagnosis and treatment of breast cancer (PCT/US97/18026).
  • ECGI proteins bind one or more anti-Mammastatin antibodies such as 7G6 (Neomarkers, Freemont, CA), and are encoded by nucleic acid sequences sharing significant homology with nucleic acid sequences encoding Mammastatin.
  • Mammastatin the ECGI proteins of the invention appear, for example, in Western blots, as doublets or triplet bands, with one major band and one or two smaller, less prominent bands. This pattern of expression was demonstrated for Mammastatin to be due to phosphorylation of the protein.
  • Mammastatin has an approximate molecular weight of 53 kilodaltons when phosphorylated at two sites. Smaller sized Mammastatin, 49 and 44 kilodaltons, correspond to one or none of the sites being phosphorylated. Phosphorylation of the Mammastatin protein is correlated with its inhibitory activity.
  • Nucleic Acid Sequences Encoding ECGI Nucleic acid sequences of the invention are defined herein as those nucleic acid sequences that encode ECGI proteins, as defined above. Nucleic acid sequences encoding ECGI proteins share significant sequence homology to nucleic acid sequences encoding Mammastatin, and hybridize to nucleic acid sequences encoding Mammastatin under conditions of high stringency.
  • Mammastatin-like epithelial cell growth inhibitors preferably have substantial identity (at least 90%, and preferably at least 95% identity) over approximately 1000 contiguous nucleotides of a nucleic acid sequence encoding Mammastatin.
  • Nucleic acids encoding Mammastatin include those DNA inserts of MammA (PCT/US97/18026, ATCC# 97451, deposited 22 February 1996); MammB (PCT/US97/27147, ATCC# PTA-2091, deposited 15 June 2000); and MammC, described herein (ATCC# PTA-2090, deposited 15 June 2000).
  • Consensus sequences determined for known Mammastatin clones are shown in the Comparative Sequence Table 5 below, and as SEQ ID NO: 1 (MarnmA); SEQ ID NO: 2 (MammB); SEQ ID NO: 3 (MammC).
  • SEQ ID NO: 4 Prostate ECGI nucleic acid sequence (SEQ ID NO: 4) is shown in Tables 1, 2, and 5.
  • ECGI can be amplified from a specific epithelial cell nucleic acid library, for example, using internal Mammastatin primers and/or by hybridization to Mammastatin under conditions of strict stringency.
  • nucleic acid sequences hybridizing to Mammastatin have been demonstrated in numerous epithelial tissues, including central nervous system, heart, small intestine, large intestine, appendix, rectum, lymphatic cells, bone marrow cells, lung and air passages, bladder, uterus, prostate, testis, ovary, liver, pancreas, adrenal gland, salivary gland, and mammary gland (See Figure 1).
  • nucleic acid sequence of a ECGI isolated from prostate cells shares greater than 95% identity to Mammastatin at the 5' half of the molecule, with little or no identity of sequence, however, at the 3' half. It is postulated that the 5' end, sharing identity with Mammastatin, includes a growth inhibitory domain of the molecule, whereas the 3' end, having little identity to Mammastatin, includes a tissue-specificity domain.
  • the invention further provides an in vitro assay for detecting active, inhibitory ECGI in patient samples, including tissues, cells, and fluids.
  • Epithelial cell cancer and advancing metastatic disease is diagnosed by correlating the presence and type of ECGI protein in a patient's sample with that of normal or cancerous human epithelial cells.
  • a patient's blood or tissue sample is analyzed for the ECGI protein, e.g., for the abundance of the ECGI protein and/or for its molecular weight forms.
  • the absence or loss of ECGI protein, particularly of the higher molecular weight, phosphorylated forms is correlated with a specific epithelial cell indicative of advancing metastatic disease.
  • ECGI Analysis of ECGI can be performed using a variety of known analytical tools and methods, including immunoassays, hybridization, PCR techniques, and the like.
  • immunoassay including ELISA, Western Blot, and dot-blot analysis of a patient's sample methods, using anti-ECGI antibodies.
  • recombinant ECGI standards are used to provide a standard curve for reliable quantitation of inhibitor levels.
  • Such immunoassays are exemplified by the dot-blot assays and Western blot assays shown in the examples below.
  • tissue samples such as tumor biopsies, are analyzed by immunohistochemistry, or by culturing a patient's tumor cells and examining the cultures for expression of ECGI.
  • an assay for the diagnosis of an epithelial cell cancer includes at least two specific antibodies: an antibody to identify the sampled tissue as epithelial tissue, such as an anti-cytokeratin antibody, and a specific anti-ECGI antibody.
  • an antibody to identify the sampled tissue as epithelial tissue such as an anti-cytokeratin antibody
  • a specific anti-ECGI antibody For example, using an immunoblot format, prostate tissue suspected of containing the prostate cancer cells is homogenized, separated on an SDS/PAGE gel, transferred to membrane, and probed with both anti-keratin and anti-prostate ECGI antibodies.
  • Isotype specific second antibodies that are conjugated to a suitable marker system such as peroxidase or alkaline phosphates are used to detect bound antibodies.
  • Membranes containing bound first and second antibodies are then developed using known colormetric or fluorometric techniques and quantitated by known methods.
  • the sample is analyzed for the size and/or phosphorylated forms of the ECGI, such as by Western Blot, using anti-ECGI antibodies.
  • a decline or absence of the high molecular weight ECGI protein form correlates with advancing cancer.
  • Diagnostic kits of the invention include ECGI protein or nucleic acid sequences encoding ECGI, for example, as controls.
  • the diagnostic kit contains one or more antibodies that bind the epithelial cell ECGI to be detected or quantified.
  • the antibodies may bind a Mammastatin-like domain (for example, 7G6), or may be tissue-specific ECGI antibodies.
  • the diagnostic kit includes one or more amplification primer or hybridization probe for the amplification and/or detection of nucleic acid sequences encoding an epithelial cell ECGI, for example, the primers used in the Examples below.
  • ECGI protein for therapeutic use is produced from epithelial cell cultures under serum free conditions or by recombinant means.
  • ECGI protein is produced in yeast or higher eucaryotic cells to achieve phosphorylation of the protein.
  • Recombinant protein is produced in host cells or by synthetic means.
  • Functional ECGI is administered to patients by known method for the administration of phosphoprotein, preferably by injection, to increase inhibitor levels in the bloodstream and increase the inhibitor's interactions with the desired epithelial.
  • the protein may be delivered to the patient by methods known in the field for delivery of phosphorylated protein agents. In general, the inhibitor is mixed with the delivery vehicle and administered by injection.
  • the dosage of inhibitor to be administered may be determined by one skilled in the art, and will vary with the type of treatment modality and extent of disease. Since Mammastatin inhibits approximately 50% of mammary cancer cell growth at a concentration of 10 ng/ml and stops growth at about 20-25 ng/ml in vitro, a useful therapeutic dosage range of ECGI is about 2.5 ⁇ g to about 250 ⁇ g administered daily dose. Preferred is approximately 125 ⁇ g daily administered dose. The aim of the administration is to result in a final body dose that is in the physiological (e.g. 15-50 ng/ml) or slightly higher range (for example, 25-75 ng/ml).
  • the preferred dosage range is about 500 ng/ml for initial treatment of metastatic disease, followed by a maintenance dosage of about 50 ng/ml.
  • an administered daily dose of about 50 ng/ml to about 750 ng/ml was sufficient to induce remission to Stage IV breast cancer patients.
  • active ECGI is a phosphorylated protein, it is anticipated that multiple doses of the inhibitor will be required to maintain growth inhibiting levels of ECGI in the patient's blood.
  • ECGI generally acts as a cytostatic agent rather than a cytocidal agent, it is expected that a maximum effect of the inhibitor will require regular maintenance of inhibitor levels in epithelial cell cancer patients.
  • the ECGI is administered in high dosages (>50 ng/ml, preferably about 50-500 ng/ml) to induce tumor regression.
  • High dosages >50 ng/ml, preferably about 50-500 ng/ml
  • Lower, maintenance doses ⁇ 50 ng/ml, preferably 20-50 ng/ml are used to prevent cancer cell growth.
  • a useful dose is that which maintains physiological levels of Mammastatin in the blood.
  • Admimstration is preferably daily, but, may be, for example, by continuous infusion, by slow release depot, or by injection once every 2-3 days. Anecdotal evidence suggests continuous administration may induce feedback inhibition, thus, a preferred administration scheme is to administer daily dose of Mammastatin for approximately 25-28 days, followed by 2-5 days without administration.
  • Assays of the present invention for detecting the presence of the functional inhibitor in human tissue and serum are useful in screening patients for epithelial cell cancer, for screening the population for those at high risk of developing epithelial cell cancer, for detecting early onset of epithelial cancer, and for monitoring patient levels of inhibitor during treatment.
  • analysis of a patient's blood ECGI may indicate a reduced amount of high molecular weight, phosphorylated prostate ECGI, as compared with a normal control or with the patient's prior prostate ECGI profile. Such a change is correlated with increased risk of prostate cancer, with early onset of prostate cancer, and with advancing metastatic prostate cancer.
  • Diagnostic assay for phosphorylated, active, 55 kD prostate ECGI preferably is by Western blot immunoassay, or ELISA using specific anti-ECGI antibodies. Screening, for example, in serum, is preferably by immunoassay, e.g., ELISA, Western blot, or dot blot assay.
  • the patient samples should be assayed within a short time of sampling (within one week), stored at 4°C (less than one year), or frozen for long term storage. Most preferably, samples are frozen until time of assay.
  • Northern blot analysis was performed on a multiple tissue expression array (Clonetech, Inc. #7775-1) to demonstrate the expression of ECGI in a variety of epithelial cell tissues.
  • a digoxin-labeled EcoRl fragment of Mammastatin, containing approximately 1800 base pairs of the 3' region ofpMammC, SEQ ID NO: 3 (approximately nucleotide 359 - end) was used as a probe.
  • the DIG- labeled Mammastatin cDNA was hybridized to the array in 10 ml easy HYB solution (Roche) for 16 hours at 65° C, with 65° C washes, anti-DIG antibody hybridization and CSPD development performed according to the manufacture's instructions.
  • the blot was then exposed to Kodak X-OMAT film for 30 minutes at room temperature.
  • the tissue plan of the multiple tissue expression array is shown in Figure IA.
  • Hybridization of the Mammastatin cDNA to the rtiRNA of the array is shown in Figure IB, and demonstrates the variety of epithelial cell tissues expressing a Mammastatin-like ECGI sequence. Specific tissues that hybridized to the
  • Mammastatin cDNA included: central nervous system, heart, small intestine, large intestine, appendix, rectum, lymphatic cells, bone marrow cells, lung and air passages, bladder, uterus, prostate, testis, ovary, liver, pancreas, adrenal gland, salivary gland, and mammary gland.
  • EXAMPLE 2 Normal Versus Cancerous Prostate Cells Normal prostate cells obtained from surgical samples and cancerous prostate cells, LnCap, obtained from the American Type Culture Collection (ATCC) were incubated and analyzed for the production of a prostate ECGI. The cells were cultured in DMEM/F12 media with 40 ⁇ M calcium, supplemented with 5% Chelex- treated horse serum, 10 ng/mL EGF, 10 ⁇ g/mL insulin, 100 ng/mL Cholera toxin and 1 ⁇ g/mL hydrocortisone for four days. Conditioned media samples were then collected and analyzed. Normal human mammary cells obtained from patient samples were incubated in the same medium and Mammastatin secreted into the culture medium was used as a control.
  • LnCap obtained from the American Type Culture Collection
  • Serum obtained from breast cancer patients was also analyzed and used as a control.
  • Sample fluids were collected and loaded by suction onto a nitrocellulose membrane on a dot blot apparatus.
  • the membranes were then probed with the anti- Mammastatin antibody 7G6, and antibody binding was detected with goat-anti mouse antibody labeled with alkaline phosphates. Color was developed with NBT/BCIP substrate system (Life Technologies).
  • the results are shown in Figure 2.
  • the anti-Mammastatin antibody recognized a protein produced by normal prostate cells but not cancerous prostate cells. This is analgous to the antibody's recognition of the mammary cell growth inhibitor, Mammastatin, produced by normal mammary cells, but not breast cancer cells.
  • This data in combination with the data from Example 1, demonstrates the production of Mammastatin-like ECGI in other epithelial cell tissues, and particularly, in prostate cells.
  • EXAMPLE 3 Differential Expression of ECGI in Prostate. Colon, and Ovary Prostate Nonnal prostate cells (Clonetech, Inc.), LnCap prostate cancer cells
  • A.T.C.C MCF7 breast cancer cells
  • A.T.C.C. MCF7 breast cancer cells
  • normal human mammary cells obtained from hospital tissue
  • Normal prostate cell lysate (D) contained a protein that was recognized by anti-Mammastatin antibody, while prostate cancer cells (LnCap) (B) and breast cancer cells (MCF7) (C) did not.
  • the protein recognized in the prostate cell lysate (D) was of a similar size to that of Mammastatin (A).
  • OvCar-ovarian cancer cells A.T.C.C
  • normal human ovarian cells patient surgery tissue
  • normal human mammary cells patient surgery tissue
  • direct lysates were prepared by removing growth media and rinsing cells with saline and SDS-PAGE sample loading buffer until viscous. Lysates were collected and separated on 10% SDS-PAGE, transferred electrophoretically onto nitrocellulose, and probed with the 7G6 anti-Mammastatin antibody.
  • the data are shown in Figure 5, where lane A contains molecular weight standards; B, OvCar- ovarian cancer cell lysate; C, normal human ovarian cell lysate; and D, normal human mammary cell lysate.
  • Figure 5 demonstrates that a Mammastatin-like ECGI protein is produced in normal human ovarian tissues and is recognized by anti-Mammastatin antibody.
  • the protein is not expressed in the ovarian cancer cells analyzed.
  • the ovarian ECGI has an approximate molecular weight of 60 kilodaltons.
  • Example 4 Differential Detection of Prostate ECGI in Blood Serum samples from three healthy male volunteers were analyzed for the presence of the prostate ECGI, and compared with that of serum from a prostate cancer patient. Serum samples were loaded at 400 microliter and 200 microliter samples in duplicate. The samples were drawn onto nitrocellulose by vacuum in a 96 well dot blot apparatus. The filters were then probed with the anti-Mammastatin antibody, 7G6, and developed with NBT/BCIP substrate. The data are shown in Figure 6.
  • NHMC normal human mammary cell cultures produced standard conditioned medium for comparison. Standards, in duplicate, contained 400, 200, 100, 50, 25, 12, and 6 microliters of NHCM medium. Serum samples from healthy adult males (A,C,D) and from an adult prostate cancer patient (B) were assayed using 400 and 200 microlites of serum sample. A prominent signal from normal serum (A,C,D) demonstrated the presence of prostate ECGI, while the prostate cancer patient's serum showed only a weak signal.
  • Normal prostate cells (Clonetech, Inc.), PC3 and LnCap prostate cancer cells (A.T.C.C.) were plated at a density of 5.0 x 10 cells per milliliter in 12 well plates in RPMI medium containing 10% fetal bovine serum. After 24 hours, the cultures were supplemented with 10% conditioned medium. Each sample was run in triplicate. Plates were allowed to incubate for six days at 37°C and 5% CO 2 , and at the end of the incubation period, cells were lysed with Cetrimide and counted using a Colter Counter. Percent inhibition was calculated by comparing treated versus non-treated wells, and the data shown in the table below.
  • Androgen-insensitive PC3 cells were not inhibited by the normal prostate cell media or by the conditioned medium obtained from normal prostate cells.
  • LnCap cells were inhibited by the addition of growth medium, with the inhibition somewhat greater by media derived from normal prostate versus media derived from cancer cells.
  • Example 6 Isolation and Characterization of Prostate ECGI DNA Nucleic acid libraries were produced from the mRNA of normal prostate cells (patient surgery tissue) and from LnCap, prostate tumor cells (A.T.C.C).
  • nucleic acid sequences in the normal and cancerous prostate cell libraries were incorporated into vectors and used to transform bacteria. Colonies of bacteria expressing the normal and cancer prostate cell nucleic acid sequences were screened by hybridization with a digoxin-labeled Mammastatin nucleic acid probe under stringent conditions, as described above.
  • Plasmids obtained from the positive colonies were purified and digested with ECO Rl and Xhol to release the CDNA inserts.
  • the digested DNA was then separated on a 1% agarose gel (see Figure 7A) and the separated DNA was subjected to Southern blot analysis using the digoxin-labeled Mammastatin fragment as a probe.
  • Figure 7 two prostate ECGI clones were isolated, each having an approximate size of 2 Kb: One clone was isolated from the normal prostate tissue library (PRN2.1) and one from the LnCap prostate tumor cell library (PRT-6).
  • PRT-6 was further characterized, and its nucleic acid sequence was determined. As shown below in Table 1, the nucleic acid sequence encoding Prostate ECGI has substantial identity to Mammastatin (greater than 90%) at the 5' end of the molecule (approximately nucleotides 15-1032 of MammC), with little or no identity at the 3' end of the molecule. These regions of similarity and distinction are shown diagrammatically in Figure 8.
  • Example 7 Isolation and Characterization of Prostate ECGI DNA Nucleic acid libraries were constructed from the mRNA or normal prostate cells (obtained from patient surgery tissue) and from LnCap prostate tumor cells (A.T.C.C). The library cDNA was used to transfer E.coli and plated out for colony hybridization. The colonies were screened with a digoxin-labeled Mammastatin C fragment generated by PCR using external PCR primers M200 and M2200. [Sequence ID NO: 5] M200: GCGCCGGCCGGGCGCGACCCG
  • the prostate ECGI sequence was analyzed against nucleic acid sequences present in GenBank. Portions of two molecules showed some similarity to domains within the prostate ECGI sequence: 28SmRNA and Hip55.
  • Hip55 is a protein that binds to hematopoetic progenitor type 1 kinase, a protein involved in the src signal transduction pathway (Ensena et al, 1999, JBC 274:33945-50).
  • Prostate GIP (1) GCACGAGATTCCCACTGTCCCTACCTACTATCCAGCGAAACCACAGCCAA
  • Prostate GIP GGGAACGGGCTTGGCGGAATCAGCGGGGAAAGAAGACCCTGTTGAGCTTG
  • Prostate GIP (101) ACTCTAGTCTGGCACGGTGAAGAGACATGAGAGGTGTAGAATAAGTGGGA
  • Prostate GIP (151) GGCCCCCGGCGCCCCCCCGGTGTCCCCGCGAGGGGCCCGGGGCGGGGTCC
  • Prostate GIP (201) GCCGGCCCTGCGGGCCGCCGGTGAAATACCACTACTCTGATCGTTTTTTC
  • Prostate GIP (251) ACTGACCCGGTGAGGCGGGGGGGCGAGCCCCGAGGGGCTCTCGCTTCTGG
  • Prostate GIP (301) CGCCAAGCGCCCGGCCGCGCGCCGGCCGGGCGCGACCCGCTCCGGGGACA
  • Prostate GIP (351) GTGCCAGGTGGGGAGTTTGACTGGGGCGGTACACCTGTCAAACGGTAACG
  • Prostate GIP (401) CAGGTGTCCTAAGGCGAGCTCAGGGAGGACAGAAACCTCCCGTGGAGCAG
  • Prostate GIP (451) AAGGGCAAAAGCTCGCTTGATCTTGATTTTCAGTACGAATACAGACCGTG
  • Prostate GIP (501) AAAGCGGGGCCTCACGATCCTTCTGACCTTTTGGGTTTTAAGCAGGAGGT
  • Prostate GIP (551) GTCAGAAAAGTTACCACAGGGATAACTGGCTTGTGGCGGCCAAGCGTTCA
  • Prostate GIP (601 TAGCGACGTCGCTTTTTGATCCTTCGATGTCGGCTCTTCCTATCATTGTG
  • Prostate GIP (651 AAGCAGAATTCACCAAGCGTTGGATTGTTCACCCACTAATAGGGAACGTG
  • Prostate GIP (701 AGCTGGGATTAGACCGTCGTGAGACAGGTTAGTTTTACCCTACTGATGAT
  • Prostate GIP (751 GTGTTGTTGCCATGGTAATCCTGCTCAGTACGAGAGGAACCGCAGGTTCA
  • Prostate GIP (801 GACATTTGGTGTATGTGCTTGGCTGAGGAGCCAATGGGGCGAAGCTACCA
  • Prostate GIP (851 TCTGTGGGATTATGACTGAACGCCTCTAAGTCAGAATCCCGCCCAGGCGG
  • Prostate GIP (901 AACGATACGGCAGCGCCGCGGAGCCTCGGTTGGCCTCGGATAGCCGGTCC
  • Prostate GIP (951 CCCGCCTGTCCCCGCCGGCGGGCCGCCCCCCC—CTCCACGCGCCCCGCG
  • Prostate GIP (999 CGCGCGGGAGGGCGCGTGCCCCGCCGCGCGCCGGGACCGGGGTCCGGTGC
  • Prostate GIP (1049 GGAGTGCCCTTCGTCCTGGGAAACGGGGCGCGGCCGGAAAGGCGGCCGCC
  • Prostate GIP (1099 CCCTCGCCCGTCACGCACCGCACGTTCGTGGGGAACCTGGCGC-TAAACC
  • Prostate GIP (1148 ACCTCCATCTCCAGTCCTCA--GCCTGGCAAGCTGAGG-AGCCCCTTCCT
  • Prostate GIP (1195 GCA—GAAG-CAGCTCACCCAACCAGAGACCCACT TTGGCAGA
  • Prostate GIP (1235 GAGCCAGCTGCTGCCATCTCAAGGCCCAGGGCAGATCTCCCTGCTGAG—
  • Prostate GIP (1283 GAGCCGGCGCC CAGCACTCCTCCATGTCTGGTGCAGGCA
  • Prostate GIP 1322 GAAGAGGAGGCTGTGTATGAG-GAACCTCCAGAGCAGGAG ACCT
  • Prostate GIP (1365 -TCTAC GAGCAGCCCCCACTGGTGCAGCAG CAAGGTGCTGGC
  • Prostate GIP (1406 TCTGAGCACATTGACCAC—CACATTCAGGGCCAGGGGCTCAGTGGGCAA
  • Prostate GIP 1454 GGGCTCTGTGCCCGTGCCCTGTACGACTACCAGGCAGCCGACGACA—CA
  • Prostate GIP (1502 GAGATCTCCTTTGA CCCCGA-GAACCTCATCACGGGCATCGAGGTGA
  • Prostate GIP (1548 TCGACGAAGGCTGGTGGCGTGGCTATGGGCCGGATGGCCATTTTGGCATG
  • Prostate GIP (1598 TTCCCTGCCAACTACGTGGAGCTCATTGAGTGAGGCTGAGGGCACATCTT
  • Prostate GIP (1648 GCCCTTCCCCTCTCAGACATGGCTTCCTTATTGCTGGAAGAGGAGGCCTG
  • Prostate GIP (1698 GGAGTTGA C ATTCAGCACTCTTCCAGGAATAGGACCCCCAGTG
  • Prostate GIP (1741 AGG-ATGAGGCCTCAGGGCTCCCTCCGGCTTGG-CAGACTCAGCCTGTCA
  • Prostate GIP (1839 CCCGACCCTCCCAGA-CAGCTTGGCTCTTGCCCCTGACAGGATACTGAGC
  • Prostate GIP (1888 CAAGCCCTGCCTGTGGCCAAGCCCTGAGTGGCCACTGCCAAGCTGCGGGG
  • Prostate GIP (2018 AAGGGGTGGTGGCCACCACTGTTTAGAATGACCCTTGGGAACAGTGAACG
  • Prostate GIP (2068 TAG AGAATTGTTTTTAGCA-GAGTTTGTGACCAAAGTCAGAGTGG—
  • Prostate GIP (2112 ATCATGGTGGTTTGGCAG--CAGGGAATTTGTCTTGTTGGAGCCT GC
  • Prostate GIP (2157 TCTGTGCTCCCCACTCCATTTCTCTGTCCCTCTGCCTGGGCTATGGGAAG
  • Prostate GIP (2207 TGGGGATGCAGATGGCCAAGCTCCCAC CCTGGGTATTCAAAAAC
  • Prostate GIP (2251 GGCAGACACAACATG-TTCCTCCACGCGGCTCACTCGATGC—CTGC
  • Prostate GIP 2295 AGGCCCCAGTGTGTGCCTCAACTGATTCTGACTTCAGGAAAAGTAAAA-A
  • Prostate GIP (2344 A AAAAAAAAAAAACTCGAGAAGCTTTGGACTTCTTCGCCA
  • 28SmRNA 201 AAAATAACAGAATACAGCACTTAATTAATTTTTTTTTTTTCCTTCGGACG pMammB (1 CGG
  • 28SmRNA 301 CACCGCAACCTCCACCTCCCGCGTTCAAGCGATTCTCCTGCCTCAGCCTC pMammB (49 GCCCCCGGCGCCCCCC—CGGTGTCCCCGCGAGGGGCCCGCGCG GGTC
  • 28Srr ⁇ RNA (351 CTGAGTAGC—-TGGGATTACAGGGAGGAGCCACCACACCCAGCTGATTTT pMammB (93 CGCCGGCCC-GCGGGC-GCCGGTGAAATACCACTACTCTGATCGTTTTTTTT
  • 28SmRNA (449 A-CTGGCGACCCCAGTGGATCTGCCCGCCCCGGCCTCCCAAAGTGCTGGG pMammB (185 TTCTGGCG—CCAAGCG CCCGGCCGCGCGCCG-GCCGGG
  • 28SmRNA (744) CAGCCTCCCAAGTAGCTGGGACTACAGGCGCCCGCCACCGTGCCCGGCTA pMammB (446) CAGGGAT—AACTGGCTTGT GGCGGCCA—AGCGTTCAAAGCGA
  • 28S RNA (794) ACTTTTTGTATTTTGAGTAGAGATGGGGTTTCACTGTGGTAGCCAGGATG pMammB (486) CGTCGCTTTTTGATCCTTCGATGTCGGCTCTTCCTATCATTGGGAAG
  • 28SmRNA (844) GTCTCGATCTCCTGACCCCGTGATCCGTCCACCTCGGCCTCCCAAA G pMammB (533) —CA-GAATTCACCAAGCGTTGGATTGTTCACCCACTAATAGGGAACGTG
  • 28SmRNA (1028 GCCAAGGCTTGGAC—CGAGGGATCCACCGGCCCTCGGCCTCCCAAAAGT pMammB (725 ACCATCTGTGGGATTATTACTGAACGCCTCTAAGTCAGAATCCCGCCCAG
  • 28SmRNA (1076 GCGGGGATGACAGGCGCGAGCCTACCGCGCC-CGGA—CCCCCCCTTTCC pMammB (775 GCGGA-ACGATACGGCAGCGCCG-CGGAGCCTCGGTTGGCCTCGGATGGC
  • 28SmRNA (1123 CCTTCCCCCGCTTGTCTTC-CCGACAGAC—AGTTTCACGGCAGAGCGTT pMammB (823 CGGTCCCCCGCCTGTCCCCGCCGGCGGGCGCCCCCCCCTCCACGCGCC
  • 28SmRNA (1216 GCTTCTG GCCTCACGGACTCTGAGCCGAGGAGTCCCCTG GTCTG pMainmB (923 GGTGCGGAGTGCCCTTCGTCCTGGGAAACGGGGCGCGGCCGGAAAGGCGG
  • 28S RNA (1260 TCTATCACAGGACCGTACACGTAAGGAGGAGAAAAATCGTAACGTTCAAA pMammB (973 CCGCCCCCTCGCCCGT-CACGCACCG-CACGTTCGTGCT CGTGCCGA
  • 28SmRNA (1360 ACCAGTCTTTTAGAAATGGCCTTAGCCCTGGTGTCCGTGCCAGTGATTCT pMammB (1065 TCT-TTATTATATAA TGAATTCTTTT—CCTTTGGGGAGATA—
  • 28SmRNA (1460 GGACGGAAGTTCCAGATGATCCGATGGGTGGGGGACTTAGGCTGCGTCCC pMaitimB (1153 TGAGAAATCTTCATACTGATTTCCATAGAGGTTGTACAAATTTACATCCC
  • 28SmRNA (1608 AGGGGGATCCCAATTCATTCCGGGCTGACACGCTCACTGGCAGGCGTCGG pMammB (1297 GGGGTA CAGGTATTTACAAGTTT-CATTAT-ACAGAC —AATTGA
  • 28SmRNA (1758 ACCGCCCCCGCC-CCCACCTCCAAGTTCCTCCCTCCCTTGTTGCCTAGGA pMammB (1435 ATCTCAGAAGAAATACATTTCTAGTAGTACCACCAGCATATATTCTACTG
  • 28SmRNA (1807 AATCGCCACTTTGACGACCGGGTCTGATTGACCTTTGATCAGGCAAAAAC pMammB (1485 AATTGGCTTTGTGATCATCATTTATACCTACTTATT AAAAC
  • 28SmRNA (2056 ATACGCCGGGCGCGGTGGCTCATGCCTGTCATCCCGTCACTTTGGGATGC pMammB (1709 TTCTGTAGGGTGGAGAA ATAGTCTTACCGTAGTAAGA
  • 28SmRNA (2254) CGCTTGAACCTGGGAAGCGGAGGTTGCAGTGAGCCGAGATTGCGCCATCG pMammB (1883) CCTGGCTCCCTKYTTATTTCCT-TTCCCTTCCTCCCACAACCCCTTTTTC
  • 28SmRNA (2304) CACTCCAGTCTGAGCAACAAGAGCGAAACTCCGTCTCAAAAATAAATACA pMammB (1932) CCCCCATTTCTTTTCTT TCTTTTTATTTGTTAATTACA
  • 28SmRNA (2404) AATTAAAATAAATAAATAAAATAAAATAAATAAATGGGCCCTGCGCGGTG pMammB (1995) ATTGAT TAGCACAAAAGGATAT AAGTAC GGG TGAGTGATA
  • 28SmRNA (2647) ACCTGGGAGGCGGAGGTTGC AGTGAGCCGAGATCGCGCCACTGCAAC pMammB (2225) GCCCAGGAGGTGGAAGCTGCAGCAGTGCGCTGAGATTGCGCCATTGCACT
  • GCCGGCCCTGCGGGCCGCCGGTGAAATACCACTACTCTTATCGTTTTTTC pMamm B (94) GCCGGCCC-GCGGGC-GCCGGTGAAATACCACTACTCTGATCGTTTTTTC pMamm C (103) GCCGGCCCTGCGGGCCGCCGGTGAAATACCACTACTCTGATCGTTTTTTC pPros (201) GCCGGCCCTGCGGGCCGCCGGTGAAATACCACTACTCTGATCGTTTTTTC
  • AAGGGCAAAAGCTCGCTTGATCTTGATTTTCAGTACGAATACAGACCGTG pMamm B (339) AAGGGCAAAA TGATCTTGATTTTCAGTACGAATACAGACCGTG pMamm C (353) AAGGGCAAAAGCTCGCTTGATCTTGATTTTCAGTACGAATACAGACCGTG pPros (451) AAGGGCAAAAGCTCGCTTGATCTTGATTTTCAGTACGAATACAGACCGTG 501 550 pMamm A (459 TAAGCGGGGCCTCACGATCCTTCTGACCTTTTGGGTTTTAAGCAGGAGGT pMamm B (382 AAAGCGGGGCCTCA-GATC-TTCTGACCTTTTGGGTTTTAAGCAGGAGGT pMamm C (403 AAAGCGGGGCCTCACGATCCTTCTGACCTTTTGGGTTTTAAGCAGGAGGT pPros (501 AAAGCGGGGCCTCACGATCCTTCTGACCTTTTGGGTTTTAAGCAGG
  • pMamm A (659 AGCTGGGTTTAGACCGTCGTGAGACAGGTTATTTTTACCCTACTGATGAT pMamm B (580 AGCTGGGTTTAGACCGTCGTGAGACAGGTT-TGTTTACCCTACTGATGAT pMamm C (603 AGCTGGGTTTAGACCGTCGTGAGACAGGTTAGTTTTACCCTACTGATGAT pPros (701 AGCTGGGATTAGACCGTCGTGAGACAGGTTAGTTTTACCCTACTGATGAT
  • pMamm A (759 GACATTTGGTGTATGTGCTTGGCTGAGGAGCCAATGGGGCGAAGCTACCA pMamm B (679 GACATTTGGTGTATGTGCTTGGCTGGGGAGCCAATGGGGCGAAGCTACCA pMamm C (703 GACATTTGGTGTATGTGCTTGGCTGAGGAGCCAATGGGGCGAAGCTACCA pPros (801 GACATTTGGTGTATGTGCTTGGCTGAGGAGCCAATGGGGCGAAGCTACCA
  • pMamm A (857 GAACGATACGGCAGCGCCGCGGAGCCTCGGTTGGCCTCGGATTAGCCGGT pMamm B (778 GAACGATACGGCAGCGCCGCGGAGCCTCGGTTGGCCTCGGATG-GCCGGT pMamm C (802 GAACGATACGGCAGCGCCGCGGAGCCTCGGTTGGCCTCGGATA-GCCGGT pPros (900 GAACGATACGGCAGCGCCGCGGAGCCTCGGTTGGCCTCGGATA-GCCGGT
  • 1101 1150 pMamm A (1057 CCCCCTCGCCCGTCACGCACCGCACGTTCGTGCT CGTGCCGAATTCG pMamm B (976 CCCCCTCGCCCGTCACGCACCGCACGTTCGTGCT CGTGCCGAATTCG pMamm C (1001 CCCCCTCGCCCGTCACGCACCGCACGTTCGTGCT CGTGCCGAATTCG pPros (1097 CCCCCTCGCCCGTCACGCACCGCACGTTCGTGGGGAACCTGGCGC-TAAA
  • 1501 1550 pMamm A 1454 TTAGGATTCAAAGTTGTATGAACAAGGACAAGTGCTCTAGGGACTTGCAA pMamm B (1371 TTAGGATTCAAAGTTGTAAGAACAAGGACAAGTGCTCTAGGGACTTGCAA pMamm C (1396 TTAGGATTCAAAGTTGTATGAACAAGGACAAGTGCTCTAGGGACTTGCAA pPros (1439 GGGCTCA GT GGGCAAGGGCTCTGTGCCCGTGCCC
  • 1651 1700 pMamm A (1604 TAAAACTAATGAAAAGGGTTTATATCAAATATACTTTAAGGTATAAAAAT pMamm B (1520 TAAAACTAATGAA ⁇ AGGGTTTATATCAAATATACTTTAAGGTAAAAA ⁇ AT pMamm C (1545 TAAAACTAATGAA ⁇ AGGGTTTATATCAAATATACTTTAAGGTATAAAAAT pPros (1554 -A ⁇ GGCTGGTGGCGTGGCTATGGGCCGGATGGCCATTTTGGCATGTTCCC

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Abstract

Des inhibiteurs de croissance des cellules épithéliales (ECGI) s'expriment de manière différe. Les protéines ECGI et la séquence d'acide nucléique codant ces dernières sont utiles dans le diagnostic et le traitement des cancers des cellules épithéliales, tels que par exemple les cancers de la prostate, des ovaires, du colon et autres.
PCT/US2000/016900 1999-06-18 2000-06-19 Inhibiteurs de croissance des cellules epitheliales WO2000078955A1 (fr)

Priority Applications (11)

Application Number Priority Date Filing Date Title
MXPA01013242A MXPA01013242A (es) 1999-06-18 2000-06-19 Inhibidores del crecimiento de celulas epiteliales.
KR1020017016294A KR20020016837A (ko) 1999-06-18 2000-06-19 상피세포 성장 억제제
EP00946824A EP1190054A1 (fr) 1999-06-18 2000-06-19 Inhibiteurs de croissance des cellules epitheliales
IL14714800A IL147148A0 (en) 1999-06-18 2000-06-19 Epithelial cell growth inhibitors
BR0012301-3A BR0012301A (pt) 1999-06-18 2000-06-19 Inibidores de crescimento de células epiteliais
AU60523/00A AU783373B2 (en) 1999-06-18 2000-06-19 Epithelial cell growth inhibitors
JP2001505697A JP2003513615A (ja) 1999-06-18 2000-06-19 上皮細胞増殖インヒビター
CA002375498A CA2375498A1 (fr) 1999-06-18 2000-06-19 Inhibiteurs de croissance des cellules epitheliales
NZ516545A NZ516545A (en) 1999-06-18 2000-06-19 Epithelial cell growth inhibitors
US10/028,952 US6939714B2 (en) 1999-06-18 2001-12-18 Epithelial cell growth inhibitors
US11/058,923 US20050169936A1 (en) 1999-06-18 2005-02-15 Epithelial cell growth inhibitors

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US13999599P 1999-06-18 1999-06-18
US60/139,995 1999-06-18

Related Child Applications (1)

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US10/028,952 Continuation US6939714B2 (en) 1999-06-18 2001-12-18 Epithelial cell growth inhibitors

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WO2000078955A1 WO2000078955A1 (fr) 2000-12-28
WO2000078955A9 true WO2000078955A9 (fr) 2002-09-06

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EP (1) EP1190054A1 (fr)
JP (1) JP2003513615A (fr)
KR (1) KR20020016837A (fr)
CN (1) CN1399680A (fr)
AU (1) AU783373B2 (fr)
BR (1) BR0012301A (fr)
CA (1) CA2375498A1 (fr)
IL (1) IL147148A0 (fr)
MX (1) MXPA01013242A (fr)
NZ (1) NZ516545A (fr)
PL (1) PL352997A1 (fr)
WO (1) WO2000078955A1 (fr)
ZA (1) ZA200200145B (fr)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6500937B1 (en) 1996-10-03 2002-12-31 University Of Michigan Nucleotide sequence encoding a mammary cell growth inhibitor
US6342533B1 (en) 1998-12-01 2002-01-29 Sepracor, Inc. Derivatives of (−)-venlafaxine and methods of preparing and using the same
AU2000257514A1 (en) * 2000-06-19 2002-01-02 The University Of Michigan Mammastatin sequence variant c
CN102787094B (zh) * 2011-05-17 2015-09-23 李晖 培养基、细胞培养用试剂盒及细胞培养方法

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* Cited by examiner, † Cited by third party
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WO1996031625A1 (fr) * 1995-04-07 1996-10-10 Cytogen Corporation Polypeptides presentant un domaine fonctionnel important, et leurs procedes d'identification et d'utilisation
WO1998014577A2 (fr) * 1996-10-03 1998-04-09 Biotherapies, Inc. Sequence nucleotique et de proteique de mammastatine et procedes d'utilisation
US6500937B1 (en) * 1996-10-03 2002-12-31 University Of Michigan Nucleotide sequence encoding a mammary cell growth inhibitor

Also Published As

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KR20020016837A (ko) 2002-03-06
AU6052300A (en) 2001-01-09
NZ516545A (en) 2004-06-25
IL147148A0 (en) 2002-08-14
MXPA01013242A (es) 2002-11-04
WO2000078955A1 (fr) 2000-12-28
EP1190054A1 (fr) 2002-03-27
JP2003513615A (ja) 2003-04-15
ZA200200145B (en) 2003-04-30
CA2375498A1 (fr) 2000-12-28
BR0012301A (pt) 2002-03-26
AU783373B2 (en) 2005-10-20
PL352997A1 (en) 2003-09-22
CN1399680A (zh) 2003-02-26

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