EPITHELIAL CELL GROWTH INHIBITORS

Abstract: Epithelial cell growth inhibitors differentially express in normal and cancerous epithelial cells. The ECGI proteins and nucleic acid sequence encoding them are useful in the diagnosis and treatment of epithelial cell cancers, for example prostate, ovarian, colon cancer, and the like.

Description: EPITHELIAL CELL GROWTH INHIBITORS Field of the Invention This invention relates to a family of epithelial cell growth inhibitors useful in the diagnosis and treatment of epithelial cell cancers. Background of the Invention 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., are 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. Although progress has been made in the diagnosis and treatment of various cancers, these diseases still result in significant mortality. The treatment of cancer is greatly enhanced by early detection. However, there are difficulties in detecting the disease in its early stages. For example, epithelial tissue- containing organs such as the prostate, ovary, and others, are not easily palpated. The detection of abnormal tumor growth in such organs is difficult without frequent screening and appropriate markers. A substantial drawback of available cancer diagnostic assays is a high rate of false positive and negative results, making the available tests less reliable than desired. For this reason, there is a great need to identify new diagnostic as well as new therapeutic agents to improve diagnosis and treatment of cancer, 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., A novel, specific, mammary cell growth inhibitor, Mammastatin, has recently been identified and characterized. Mammastatin has been expressed from variant clones, MammA (PCT/US97/18026, ATCC# 97451, deposited 22 February 1996); MammB (PCT/US97/27147, ATCC# , deposited 15 June 2000); and MammC, described in copending PCT application No. PCT/US00/ , filed on even date herewith (ATCC# , 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 inhibitors 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. Like Mammastatin, the newly discovered family of epithelial cell growth inhibitors (ECGI) 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. Summary of the Invention A family of epithelial cell growth inhibitors (ECGI) have now been identified in a number of different epithelial cells. These ECGI are differentially expressed in normal epithelial cells, but not in epithelial cancer cells. As shown in the Examples below, Mammastatin-like ECGI proteins have been discovered in a variety of epithelial cell tissues, including prostate, colon, ovary, lung, spleen, testis, thymus, and others. 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# , deposited 15 June 2000), as described in the Examples below. Because 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. Brief Description of the Figures Figure 1 A 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 mRNA from a variety of tissues according to the plan shown in Figure 1A. 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. Detailed Description of the Invention Proteins of the invention: "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. Studies reported in the Examples below demonstrate the differential expression of ECGI proteins in normal epithelial cell tissues, but not in cancerous epithelial cell tissues, including breast, prostate, ovary, and colon. Like 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. Western blots of ECGI probed with the anti-Mammastatin antibody 7G6, demonstrate the approximate size of ECGI produced by various epithelial cell tissues. As shown more fully in the Examples below (see, for example, Figures 4-5), ECGI from prostate cells migrates in a Western blot to approximately 55 kilodaltons, with less prominent, smaller bands at 51 and 46 kilodaltons suggestive of phosphorylated forms similar to the pattern seen for Mammastatin. ECGI from colon cells migrates to approximately 50 KD, with less prominent bands at approximately 47 and 43 kilodaltons. ECGI from ovarian cells migrates to approximately 60 kilodaltons. 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# , deposited 15 June 2000); and MammC, described herein (ATCC# , 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 (MammA); SEQ ID NO: 2 (MammB); SEQ ID NO: 3 (MammC). 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. As shown more fully in the Examples below, 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). The nucleic acid sequence of a ECGI isolated from prostate cells, for example, 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. Diagnostic Methods 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. As discussed below, 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. Analysis of ECGI can be performed using a variety of known analytical tools and methods, including immunoassays, hybridization, PCR techniques, and the like. Preferred are immunoassay, including ELISA, Western Blot, and dot-blot analysis of a patient's sample methods, using anti-ECGI antibodies. Preferably, 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. In an alternative preferred embodiment of the invention, 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. In a particularly preferred embodiment, 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. 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. In the most preferred embodiment, 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. Optionally, 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. Alternatively, 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. Therapeutic Use ECGI protein for therapeutic use is produced from epithelial cell cultures under serum free conditions or by recombinant means. Preferably, 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 μg to about 250 μg administered daily dose. Preferred is approximately 125 μg 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). For clinical use, 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. In clinical studies using Mammastatin, 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. Since 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. Also, since 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. In its preferred use, the ECGI is administered in high dosages (> 50 ng/ml, preferably about 50-500 ng/ml) to induce tumor regression. Lower, maintenance doses (<50 ng/ml, preferably 20-50 ng/ml) are used to prevent cancer cell growth. Clinical experience with administered Mammastatin in Stage IN breast cancer patients indicates a useful dose is that which maintains physiological levels of Mammastatin in the blood. Administration 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. Diagnostic Assay 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. For example, analysis of a patient's blood ECGI, for example, 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. For best results, 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. EXAMPLES The invention may be better understood by reference to the following Examples, which are not intended to limit the invention in any way. EXAMPLE 1 Multiple Tissue Expression of ECGI 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 of pMammC, 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 1 A. Hybridization of the Mammastatin cDNA to the mRNA 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 μM calcium, supplemented with 5% Chelex-treated horse serum, 10 ng/mL EGF, 10 μg/mL insulin, 100 ng/mL Cholera toxin and 1 μg/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. 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 Normal prostate cells (Clonetech, Inc.), LnCap prostate cancer cells (A.T.C.C), MCF7 breast cancer cells (A.T.C.C.) and normal human mammary cells (obtained from hospital tissue) were incubated as described above for Example 2. After at least 48 hours incubation, cells were lysed in sample loading buffer and analyzed for the presence of ECGI by Western blot, using the anti-Mammastatin antibody, 7G6 as a probe. Normal human mammary cell protein (NHMC) lysate (1 mg/ml) was used as a Mammastatin control (A). The data are shown in Figure 3. 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). Colon and Prostate Normal prostate cells (Clonetech, Inc.), LnCap prostate cancer cells (A.T.C.C), Sw 948 colon cancer cells (A.T.C.C), and normal colon epithelial cells (obtained from patient surgery tissue) were incubated as described above for Example 2. Cell lysates were prepared in sample loading buffer and analyzed for expression of ECGI by Western blot, using the anti- Mammastatin antibody, 7G6 as a probe. As shown in Figure 4, normal prostate (A) and normal colon (C) epithelial cells expressed a protein that was recognized by the anti-Mammastatin antibody, while cancer cells from these tissues did not (B,D). The differential expression of protein is similar to that demonstrated for Mammastatin in breast tissue. In addition, the pattern of bands shown in the Western blot for normal prostate and colon tissues is similar to the Phosphorylation pattern demonstrated for Mammastatin produced in normal human mammary cells. A larger prominent band is shown together with two smaller, fainter bands. This pattern has been correlated with Phosphorylation of Mammastatin. Prostate ECGI is shown in the Western blot analysis (Figure 4) to have an approximate molecular weight of 51 kilodaltons; Colon ECGI is shown to have an approximate molecular weight of 50 kilodaltons. Ovary OvCar-ovarian cancer cells (A.T.C.C), normal human ovarian cells (patient surgery tissue) and normal human mammary cells (patient surgery tissue) were incubated as described above for Example 2. After an incubation period of at least 48 hours, 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. Normal human mammary cell (NHMC) 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. Example 5 Inhibitory Activity of Prostate ECGI 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 4 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. In contrast, 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). The 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. The positive colonies were selected and grown in LB broth. 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. As shown in Figure 7 below, 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 [Sequence ID NO: 6] M2200: GCAATCTCAGCGCACTGCTGC Bacterial colonies expressing prostate ECGI clones were hybridized to the labeled Mammastatin probe under strict hybridization conditions, as described above. Example 8 Homology of Prostate ECGI 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. 28SmRNA homology has been identified in many gene sequences with importance in growth regulation (Hu et al., 1999, PNAS 96:1339-1344; Mauro et al., 1997, PNAS 94:422- 427). 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). Using the open reading frame known for Hip55, a putative amino acid sequence was deducted for the prostate clone. As shown below in Table 3, the translation includes several internal stop codons. Also using the Hip55 ORF, a putative amino acid sequence was deduced for MammB and MammC sequences, shown in Tables 4 and 5.

Claims: WE CLAIM: 1. A Mammastatin-like epithelial cell growth inhibitor having substantial sequence identity to Mammastatin over at least one third of the molecule. 2. The inhibitor of claim 1, wherein the epithelial cells are 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. 3. A nucleic acid sequence encoding an epithelial-cell growth inhibitor, which inhibitor inhibits the growth of non-mammary epithelial cells, and wherein the nucleic acid sequence can be amplified from normal non-mammary epithelial cells by internal PCR primers derived from Mammastatin. 4. The nucleic acid sequence of claim 3, wherein the epithelial cells are colon, ovary, prostate, spleen, testis, or thymus cells. 5. A method for detecting epithelial cell cancer, comprising: analyzing body fluid for the presence or amount of the protein of claim 1 or nucleic acid sequence of claim 3 ; and correlating a reduction or absence of said protein or said nucleic acid sequence with epithelial cell cancer. 6. A method for treating epithelial cell cancer in a patient comprising: administering to the patient the protein of claim 1 or nucleic acid sequence of claim 3. 7. A diagnostic kit comprising: the protein of claim 1 and/or the nucleic acid sequence of claim 4; and optionally an antibody that specifically binds the protein of claim 1.

Inventors: ERVIN PAUL R JR, ERVIN, PAUL, R., JR.
Applicants: BIOTHERAPIES INC, ERVIN PAUL R JR, BIOTHERAPIES, INC., ERVIN, PAUL, R., JR.
Application year: 2000
Main classification: C12N15
IPC codes: A61K38, A61K48, A61P35, C07K14, C12N15, C12Q1, G01N33