CA2375498A1 - Epithelial cell growth inhibitors - Google Patents

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); Mamma (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 Fi es Figure 1A is a schematic diagram of an mRNA test panel showing locations of specific tissue mRNAs for analysis.
Figure 1B 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); Mamma (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 (Mamma); 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 WO 00/78955 PCT/~JS00/16900 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 ~.g to about 250 pg administered daily dose. Preferred is approximately 125 p.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).
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 IV 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.

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-GMAT film for 30 minutes at room temperature.
The tissue plan of the multiple tissue expression array is shown in Figure 1A.
Hybridization of the Mammastatin cDNA to the mRNA of the array is shown in Figure 1B, 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.

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 pM calcium, supplemented with 5 % Chelex-treated horse serum, 10 ng/mL
EGF, 10 pg/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.
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.

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' cells per milliliter in 12 well plates in 12PMI
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 % COz, 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.
Cell Type % Inhibition by % Inhibition Normal Prostate by medium Prostate Tumor medium LnCap #1 22.5 + /- 3.3 8.3 + /- 0.4 LnCap #2 22.7 + /- 0.6 16.7 + /- 15.8 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 R1 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 Mamma and MammC sequences, shown in Tables 4 and 5.

EPO - DO ~
18. 1 Q 2000 SEQUENCE LISTING
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<120> EPITHELIAL CELL GROWTH INHIBITORS
<130> 4273.3WOU1 <140> PCT/US00/16900 <141> 2000-06-19 <150> 607139;995 <151> 1999-06-18 <160> 10 <170> Patentln Ver. 2.1 <210> 1 <211> 2418 <212> DNA
<213> Homo sapiens <400> 1 tggggctcca ccccggtggc ggccgctcta gaactagtgg atcccccggg ctgcaggaat 60 tcggcacgag cacggtgaag agacatgaga ggtgtagaat ccgtgqgagg cccccggcgc 120 ccccccggtg tccccgcgag gggcccgggg cggggtccgccggccctgcg ggccgccggt 180 gaaataccac tactcttatc gttttttcac tgacccggtc gagcgggggg gcgagccccg 290 aggggctctc gcttctggcg ccaagcgccc ggccgcgcgc cggccgggcg cgacccgctc 300 cggggacaqt gccaggtggg gagtttgact ggggcggtac acctgtcaaa cggtaacgca 360 ggtgtcctaa ggcgagctca qggaggacag aaacctcceg tggagcaqaa gggcaaaagc 420 tcgcttgatc ttgattttca gtacgaatac agaccgtgta agcggggcct cacgatcctt 480 ctgacctttt gggttttaag caggaggtgt cagaaaagtt accacaggga taactggctt 540 gtgqcggcca agcgttcatt aggacgtcgc tttttgatcc ttcgatgtcg gctcttccta 600 tcattgtgta gcagaattca ccaagcgttg gattgttcac ccactaatag ggaacgtgag 660 ctgggtttag accgtcgtga gacaggttat ttttacccta ctgatga.ttg tttgttgcca 720 tggttatcc.t gctcagtacg agaggaaccg caggttcaga eatttggtgt atgtgcttgg,780 ctgaggagcc aatggggcga agctaccatc tgtgggatta tgactgacgc tctaagtcat 890 gaatcccgcc caggcggaac gatacggcag cgccgcggag cctcggttgg cctcggatta 900 gccggtcccc cgcctgtccc cgccggcggg ccgccccccc ccctccacgc gccccgcgcg 960 cgcgggaggg cgcgtgcccc gccgcgcgcc gggaccgggg tccggtgcgg agtgcccttc:-1020 gtcctgggaa acgqggcqcg gccggaaagg cggccgcccc ctcgcccgtc acgcaccgca 1080 cgttcgtgct cgtgccgaat tcggcacgag tgcacccatt cacaatatac atacaagtgc 1140 atgtatcttt atgatataat gaattctttt cctttgggta gatatccagt agtgggattg 1200 ctagatcacc tggtagttct atttctggtt tatttagaaa tcttcatact gatttccata 1260 gaggttgtac aaatttacat ccctaccaaa gtgatttttt taaatatgaa agaatggtct 1320 ggagaaatgc ccctcattag tatccccctt ttacctctct actgcagaat gacttcaagg 1380 ggtacaggta tttacaagtt tcattataca gacaaattga atattgaaat tttctgcata 1940 ~f ~ry~e 20'-09 2E~01~ , 'e,~~~~~~ ~ ~~ ~ .~ , ~ '= _ 5~wh-L~R.23"fineaaik. Fti~:~.W?~' .~44.ei~:.we~r.G~i~.uxu " _.. ... t'1 .. .... ,.::.! ".,.v..";,,. ....._.

i~~_ ..rs~: . 'r,:.. , agaggcacag attttaggat tcaaagttgt atgaacaagg acaagtgctc tagggacttg 1500 caaagctgga attggaaatc tcagatgaaa tacatttcta gtagtaccac cagcatatat 1560 tctactgaat tggcttttgt gatcatcatt aatacctact att:aaaact-aatgaaaagg 1620 gtttatatca aatatacttt aaggtataaa aatcasatta taggta agc tgttttcttt 1680 agcattttaa tttcaaaaca taaaatagct acegtctatt gqgcatttat actgtacgag 1790 acactgtgtt tgtcacattt caaaaatgtt'ctcatggtaa tgttcacaat aattctgtcg 1800 ggtgagaaaa tagtcttacc gtagtaagac tattcagtaa aacgaaacct ctgaaccttg 1860 gagttcaact tgcgcaaagt tagtaacagg actaggactt gaacctgaaC catcacactc 1920 gagatctctc cataccacac tgctagcaca tgtgcctgtc atcttattcc tggctccctt 1980 ttttatttcc tttcccttcc tcccacaacc cctttttccc cccatttctt ctt~ctttt 2090 tatttqttaa ttacataact aatacatgtt tatgagaaca attgatatag cacaaaagga 2100 tataaagtac gggggagtga tagctcatcc ctgtaatcct agcactttgg aaggccaagg:2160 caggcagatc actttgagtc cagagttcga gaccagcctg ggcaacatgg 'tgaaaccctg 2220 tctctacaaa aaaatacaaa aaatttagcc gggcgtgctg gcacagacct gtagtctcag.2280 ctactctgag ggctgaggtg ggaagattga ttgagcccag gaggtggaag ctgcagcagt 2390 gcgctgagat tgcgccattg cactccagcc tgggtgagag agagagaccc tgtctccaaa 2900 aaaaaaaaaa aaaaaaaa 2418 <210> 2 <211> 2326 <212> DNA
<213> Homo sapiens <400> 2-- . _ _ _ cggcacgagc acggtgaaga gacatgagag gtgtagaata agtgggaggc ccccggcgcc 60 cccccggtgt ccccgcgagg ggcccgcggg tccgccggcc cgcgggcgcc ggtgaaatac 120 cactactctg atcgtttttt cactgacccg gtgaggcggg gggcgagccc cgaggggctc X80 tcgcttctgg-Cgccaagcgc ccggccgcgc gccggccggg cgcgaccCgc tccggggaca 240 gtgccagtgg ggagtttgac tggggcggta cacctgtcaa acggtaacgc aggtgtccta 300 aggcgagctc agggaggaca aaacctcccg tggagcagaa gggcaaaatg atcttgattt 360 tcagtacgaa tacagaccgt gaaagcgggg cctcagatct tctgaccttt tgggttttaa,920 gcaggaggtg tcagaaaagt taccacaggg ataactggct tgtggcggcc aagcgttcaa 980 agcgacgtcg ctttttgatc cttegatgtc ggctcttcct atcattggga agcagaattc 590 accaagcgtt ggattgttca-cccactaata gggaacgtga gctgggtt.ta gaccgtcgtg .600 agacaggttt gtttecccta ctgatgatgt gttgttgcca tggtaatcct gctcagtacg 660 agaggaaceg caggttcaga -catttggtgt atgtgcttgg ctggggagcc aatggggcga 720 agctaccatc tgtgggatta ttactgaacg cctctaagtc agaatcccgc ccaggcggaa 780 cgatacggca gcgccgcgga gcctcggttg gcctcggatg gccggtcccc cgcctgtccc 890 cgccggcggg cgcccceccc cctccacgcg ccccgcgcgc gcgggagggc gcgtgccccg 900 ccgcgcgccg ggaccggggt ccggtgcgga gtgcccttcg tcctgggaaa cggggcgcgg 960 ccggaaaggc ggccgccccc tcgcccgtca cgcaccgcac gttcgtgctc gtgccgaatt 1020 cggcacgagt agcaccattc acaatagaca tacaagtgca tgtatcttta ttatataatg 1080 aattcttttc ctttggggag atatccagta gtgggabtgc tagatcacct ggtagttcta 1190 tttctggttt attgagaaat cttcatactg atttccatag aggttgtaca aatttacatc 1200 cctaccaagt gattttttta aatatgaaag aatggtctgg agaaatgccc ctcattagta 1260 tccccctttt acctctctac tgcagaatga cttcaagggg tacaggtatt tacaagtttc 1320 attatacaga caaattgaat attgaaattt ctgcattaga ggcacagatt ttaggattca 1380 ''6 ' "'11f" ' CA 02375498 2001-12-17 aagttgtaag aacaaggaca agtgctctag ggacttgcaa agctggaatt ggaaatctca 1440 gaagaaatac atttctagta gtaccaccag catatattct actgaattgg ctttgtgatc 1500 atcatttata cctacttatt aaaactaatg aaaagggttt atatcaaata tactttaagg 1560 taaaaaaatc aaattatagg aaaagctgtt ttcttttgca ttttaatttcaaaacaaaaa 1620 atagctaccg tctattgggc atttatactg taccagacac tgtgtttgtc acatttcaaa 1680 aatgttctca tggtaatgtt cacaataatt ctgtagggtg gagaaatagt cttaccqtag 1740 taagactatt cagaaacgaa acctcegaac cttggagttc aacttgcgca aagttagtaa 1800 caggactagg act.tgaacct gaaccatcac actccagatc tctccatacc acactgctag 1860 cacatg.tgcc tgtcatctta ttcctggctc cctkyttatt tcctttccct tcctcccaca 1920 accccttttt ccccccattt cttttctttc tttttatttg ttaattacat aactaataca 1980 tgtttatcag aacaattgat atagcacaaa agqatataaa gtacgggtga gtgatagctc 2040 atccctgtaa tctagcactt tggaaggcca aggcag_gcag atcacttgat ccaz~agttcg 2100 agaccagcct gggcaacatg gtgaaaccct gtctctacaa aaaaatacaa aaatttagcc.2160 gggcgtgctg gcacacacct gtagtctcag ctactctgag ggctgaggtg ggaagattga -2220 ttgagcccag gaggtggaag ctgcagcagt gcgctgagat tgcgccattg cactccagcc :2280 tgggtgagag agagagaccc tgtcttcaaa aaaaaaaaaa aaaaaa 2326 <210> 3 <211> 2355 <212> DNA
<213> Homo Sapiens <400> 3 gaattcggca cgagcacqgt_gaagagacat gaqaggtgta gaataagtgg gaggcccccg 60 gcgccccccc ggtgtccccg cgaggggccc ggggcggggt ccgccggcec tgcgggccgc 120 cqgtgaaata.ccactactct gatcgttttt tcactgaccc ggtgagqcqg:,gggggcgagc-180 cccgaggggc tctcgcttct ggcgccaagc gcccggccgc_gcgccggccg:'g_gcgcqaccc 240 gctccgggga cagtgccagg tggggagttt gactggggcg gtacacctqt caaacggtaa 300 cgcaggtgtc ctasgqcgag ctcagggagg, acagaaacct cccgtggagc agaaqggcaa 360 aagctcgctt gatcttgatt ttcagtacga atacagaccq tgaaagcggg gcctcacgat 420 ccttctgacc ttttgggttt taagcaggag",gtgtcagaaa agttaccaca.gggataactg 480 gcttgtggcg gccaagcgtt cat,agcgacg tcgctttttg atccttcgat gtcggctctt 590 cctatcattg tgaagcagaa ttcaccaagc gttggattgt tcacccacta ataq.ggaacg, 600 tgagctgggt ttagaccgtc gtgagacagg ttagttttaa cctactgatg atgtgttgtt 660 gccatggtaa tcctgctcag tacgagagga accgcaggtt cagacatttg gtgtatgtgc 720 ttqgctgagg agccaatggg gcgaa'gctac catctgtggg attatgactg aacgcctcta 780 agtcagaatc ccgcccagqc ggaacgatac ggcagcgccg cggagcctcg.gttggcctcg 840 gatagccggt cccecgcctg tccccgccgg'cgggccgccc ccccccetcc acgcgccccg 900 cgcgcgcggg agggcgcgtg ccccgccgcg cgccgggacc ggggtccggt gcggagtgcc 960 cttcgtcctg ggaaacgggg cgcggccgga aaggcggccg ccccctcgcc cgtcacgcac 1020 cgcacgttcg tgctcgtgcc gaattcggca cgagtagcac cattcacaat agacatacaa 1080 gtgcatgtat ctttatgata taatgaattc ttttcctttg ggtagatatc cagtagtggg 1190 attgctagat cacctggtag ttctatttct ggtttattga gaaatcttca tactgatttc 1200 catagaggtt gtacaaattt acatccctac caagtgattt ttttaaatat gaaagaatgg 1260 tctggagaaa tgcccctcat tagtatcccc cttttacctc tctactgcag aatgacttca 1320 aggggtacag gtatttacaa gtttcattat acagacaaat tgaatattga aatttctgca 1380 taagaggcac aqattttagg attcaaagtt gtatgaacaa ggacaagtgc tctagggact 1440 a e~ ;~. a~~wy . n .
~~ r, .~~ ' ~Prmted:2~-09 2~~01 ~~ a ~~ _; ~t ~ ~ ~.~r~. .

.~ ~'~-.R~o- t ; , - _ . ' .

tgcaaagctg gaattggaaa °ctcagatga aatacat tc tagtagtacc accagcatat 1500 attctactga attggctttg tgatcattat taatacctac ttattaaaac taatgaaaa'g 1560 ggtttatatc aaatatactt taaggtataaaaatcaaatt.ataggtaaag ctgttttctt 1620 tagcatttta atttcaaaac ataaaatagc taccgtctat tgggcattta tactgtacca 1680 gacactgtgt ttgtcacatt tcaaaaatgt tctcatggta atgttcacaa taattctgta 1740 gggtgagaaa tagtcttacc gtagtaagac tattca9taa acgaaacctc tgaaccttgg 1800 agttcaactt qcgcaaagtt agtaacagga ctaggacttg aacctgaacc atcacactcc 1860 agatctctcc ataccacact gctagcacat gtgcctgtca tcttatt~cct-ggctcctgtt 1920 atttcccttt ttatttcctt tcccttcctc ccacaacccc tttttccccc catttctttt 1980 ctttcttttt aattgttaat tacataacta atacatgctt atcagaacaa ttgatatagc 2090 acaaaaggat ataaagtaeg ggtgagtgat agctcatccc tgtaatccta gcactttgga 2100 aggccaaggc aggcagatca cttgagtcca gagttcgaga ccagcctggg caacatggtg 2160 aaaccctgtc tctacaaaaa aatacaaaaa tttagccggg cgtgctggca cacacctgta 2220 gtctcagcta ctctgagggc tgaggtggga agattgattg-agcccaggag gtggaagctg'2280 cagcagtgcg ctgagattgc gccattgcac tccagcctgg gtgagagaga gagaccctgt 2340 sctcaaaaaaa aaaaa 2355 <210> 9 <211> 2333 <212> DNA
.<213> Homo Sapiens <900> 9 gcacgagatt cccactgtcc ctacctatta tccagcgaaa ccacagccaa gggaacgggc--60 ttggcqgaat cagcggggaa agaagaccct gttgagcttg ggcccccggc gcccccccgg 120 tgtccccgcg aggggcccgg ggcggggtcc gccggccctg cgggccgccg gtgaaatacc 180 actactctga tcgttttttc actgacccgg tgaggcgggg gggcgagccc cgaggggctc 240 tcgcttctgg cgccaagcgc ccggccgcgc gccggccggg cgcgacccgc tccggggaca 300 gtgccaggtg gggagtttga ctggggcggt acacctgtca aacggtaacg caggtgtcct 360 aaggcgagct cagggaggac agaaacctcc cgtggagcag aagggcaaaa gctcgcttga 420 tcttgatttt cagtacgaat acagaccgtg aaagcggggc ctcacgatcc ttctgacctt 980 ttgggtttta agcaggaggt gtcagaaaag ttaccacagg gataactggc ttgtggcggc 540 caagcgttca tagcgacgtc gctttttgat ccttcgatgt cggctcttcc tatcattgtg 600 aagcagaatt caccaagcgt tggattgttc acccactaat agggaacqtg agctgggatt 660 agaccgtcgt gagacaggtt agttttaccc tactgatgat gtgttgttgc catggtaatc 720 ctgctcagta cgagaggaac cgcaggttca gacatttggt gtatgtgctt ggctgaqgag 7B0 ccaatggggc gaagctacca tctgtgggat tatgactgaa cgcctctaag tcagaatccc 890 gcccaggcgg aacgatacgg cagcgccgcg gagcctcggt tggcctcgga tagccggtcc 900 cccgcctgtc cccgccggcg ggccgccccc ccctccacgc gccccgcgcg cgcgqgaggg 960 cgcgtgcccc gccgcgcgcc gggaccgggg tccggtgcgg agtgcccttc gtcctgggaa 1020 acggggcgcg gccggaaagg cggccgcccc ctcgcccgtc acgcaccgca cgttcgtggg 1080 gaacctggcg ctaaaccacc tccatctcca gtcctcagcc tggcaagctg aggagcccct 1140 tcctgcagaa gcagctcacc caaccagaga cccactttgg cagagagcca gctgctgcca 1200 tctcaaggcc cagggcagat ctccctgctg aggagccggc gcccagcact cctccatgtc 1260 tggtgcaggc agaagaggag gctgtgtatg aggaacctcc agagcaggag accttctacg 1320 agcagccccc actggtgcag cagcaaggtg ctggctctga gcacattgac caccacattc 1380 agggccaggg gctcagtggg caagggctct gtgcccgtgc cctgtacgac taccaggcag 1940 .Printed 20=09 X00'1 ,~~~~j~~~~ ,r . ,, ..: _ ... . . . . . ,: r. _ .. a - ~~r_..,~;~ ~.2..: .. :_._ ccgacgacac agagatctcc tttgaccccg agaacctcat cacgggcatc gaggtgatcg 1500 acgaaggctg gtggcgtggc tatgggccgg atggccattt tggcatgttc cctgccaact 1560 acgtggagct cattgagtga ggctqagggc acatcttgcc cttcccctct cagacatggc 1620 ttccttattg ctggaagagg aggcctggga gttgacattc agcactcttc caggaatagg 1680 acccccagtg aggatgaggc ctcagggctc cctccggctt ggcagactca gcctgtcacc 1790 ccaaatgcag caatggcctg gtgattccca cacatccttc ctgcatcccc cgaccctccc 1800 agacagcttg gctcttgccc ctgacaggat actgagccaa gccctgcctg tggccaagcc 1860 ctgagtggcc actgccaagc Cgcggggaag ggtcctgagc aggggcatct gggagqctct 1920 ggctgccttc tgcatttatt tgcctttttt ctttttctct tgcttctaag gggtggtggc 1980 caccactgtt tagaatgacc cttgggaaca gtgaacgtag agaattgttt ttagcagagt 2090 ttgtgaccaa agtcagagtg gatcatggtg gtttggcagc agggaatttg tcttgttgga 2100 gcctgctctg tgctccccac tccatttctc tgtccctctg cctgggctat gggaagtggg 2160 gatgcagatg gccaagctcc caccctgggt attcaaaaac ggcagacaca acatgttcct 2220 ccacgcqgct cactcgatgc ctgcaggccc cagtgtgtgc ctcaactgat tctgacttca 2280 ggaaaagtaa aaaaaaaaaa aaaaaactcg agaagctttg gacttcttcg cca 2333 <210> 5 <211> 21 <212>- DNA
<213> Homo Sapiens <900> 5 gcgccggccg ggcgcgaccc g 21 <2i0> 6 <211> 2T
<212> DNA
<213> Homo sapiens <400> 6 gcaatctcag cgcactgctg c 21 <210> 7 <211> 2843 <212> DNA
<213> Homo Sapiens <400> 7 ctttgggagg ccgaqgccgt aggatccctc gaggaatcgc ctaaccctgg ggaggttgag 60 gttgcagtga gtgagccata gttgtgtcac tgtgctccag tctgggcgaa agacagaatg 120 aggccctgcc acaggcaggc aggcaggcag gcaggcagaa agacaacagc tgtattatgt 180 tcttctcagg gtaggaagca aaaataacag aatacagcac ttaattaatt tttttttttt 290 ccttcggacg gagtttcact cttggtgccc acgctggagt gcagtggcac catctcggct 300 caccgcaacc tccacctccc gcgttcaagc gattctcctg cctcagcctc ctgagtagct 360 gggattacag ggaggagcca ccacacccag ctgattttgt attgttagta gagacggcat 920 ~.a~~.=:
I
CI ~2C~.~~ ~ ~~d1.: ~ a ~ ~ ~; ~f~'_ -,b _,. , _ _. . . ., .......... .. ,. ...." .. _ ...._ m~"'~°-~W°*~~"'~~°'~' "°'"'q"'~u~""°"~

r,k_..:~

ttctccatgt gggtcaggct.ggtctcgaac tggcgacccc agtggatctg cccgccccgg 980 cctcccaaaq tgctggggtg-acaggcgtga gccatcgtga ctggccggct acgtttattt:540 atttattttt ttaattattt tacttttttt tagttttcca ttttaatcta tttatttat.t 600 tacatttatt tatttattta tttatttact tatttattta ttttcgagac agactctcgc.660 tctgctgccc aggctggagt gcagcggcgt gatctcggct cactgcaacg :tccgcctccc 720 gggttcacgc cattctcctg cctcagcctc ccaagtagcG.gggactacag-gcgcccgcca 780 ccgtgcccgg ctaacttttt gtattttgag tagagatggg gtttcactgt ggtagccagg,890 atggtctcga,tctcctgacc,ccgtgatccg tccacctcgg cctcccaaag tgctgggatg :900 acaggcgtga gccaccggct ccggcctatt tatctattta ttaactttga gtccaggtta 960 tgaaaccagt tagtttttgt:aatttttttt tttttttttt ttttttgaga,cgaggtttca 1D20 ccgtgttgcc aaggcttgga ccgagggatc caccggccct cggcctecca aaaqtgcggg 1080 gatgacaggc:,gcgagcctac cgcgcccgga cccccccttt ccccttcccc,-cgcttgtctt 1190 cccgacagac agtttcacgg cagagcgttt ggctggcgtg cttaaactca ttctaaatag 1200 aaatttggga cgtcagcttc tggcctcacg gactctgagc cgaggagtcc cctggtctgt 1260 ctatcacagg accgtacacg taaggaggag aaaaatcgta acgttcaaag tcagtcattt 1320 tgtgatacag aaatacacgg attcacccaa aacacagaaa ccagtctttt agaaatggcc 1380 ttagccctgg tgtccgtgcc agtgattctt ttcggtttgg accttgactg agaggattcc 1940 cagtcggtct ctcgtctctg gacggaagtt ccagatgatc cgatgggtgg gggacttagg.1500 ctgcgtcccc ccaggagccc tggtcgatta gttgtgggga tcgccttgga gggcgcggtg 1560 acccactgtg ctgtgggagc ctccatcctt ccccccaccc cctccccagg gggatcccaa 1620 ttcattccgg gctgacacgc tcactggcag gcgtcgggca tcacctagcg gtcactgtta 1680 ctctgaaaac ggaggcctca cagaggaagg gagcaccagg ccgcctgcgc acagcctggg 1740 gcaactgtgt cttctccacc gcccccgccc ccacctccaa gttcctccct cccttgttgc 1800 ctaggaaatc gccactttga cgaccgggtc tgattgacct ttgatcaggc aaaaacgaac 1860 aaacagataa ataaataaaa taacacaaaa gtaactaact aaataaaata agtcaataca 1920 acccattaca atacaataag atacgatacg ataggatgcg ataggatacg ataggataca 1980 atacaatagg atacgataca atacaataca atacaataca atacaataca atacaataca 2090 atacaataca atacaatacg ccgggcgcgg tggctcatgc ctgtcatccc gtcactttgg 2100 gatgccgagg tggacgcatc acctgaagtc gggagttgga gacaagcccg accaacatgg 2160 agaaatcccg tctcaattga aaatacaaaa ctagccgggc gcggtggcac atgcctataa:2220 tcccagctgc taggaaggct gaggcaggag aatcgcttga acctgggaag cggaggttgc 2280 agtgagccga gattgcgcca tcgcactcca gtctgagcaa caagagcgaa actccgtctc 2390 aaaaataaat acataaataa atacatacat acatacatac atacatacat acatacatac 2400 ataaattaaa ataaataaat aaaataaaat aaataaatgg gccctgcgcg gtggctcaag 2460 cctgtcatcc cctcactttg ggaggccaag gccggtggat caagaggcgg tcagaccaac 2520 agggccagta tggtgaaacc ccgtctctac tcacaataca caacattagc cgggcgctgt 2580 gctgtgctgt actgtctgta atcccagcta ctcgggaggc cgagctgagg caggagaatc 2690 gcttgaacct gggaggcgga ggttgcagtg agccgagatc gcgccactgc aacccagcct 2700 gggcgacaga gcgagactcc gtctccaaaa aatgaaaatg aaaatgaaac gcaacaaaat 2760 aattaaaaag tgagtttctg gggaaaaaga agaaaagaaa aaagaaaaaa acaacaaaac 2820 agaacaaccc caccgtgaca tac 2893 <210> 8 <211> 1331 <212> DNA
<213> Homo Sapiens ..., , ~ ,. , 's: # _ . .., .. n ., .. y.,~. ° ' ~,~" ','~,y~ ~,. ...
k~::'~G

CA 02375498 2001-12-17 "f <900> 8 atggcggcga acctgagccg gaacgggcca gcgctgcaag aggcctacgt gcgggtggtc 60 accgagaagt ccccgaccga ctgggctctc tttacctatg aaggcaacag caatgacatc 120 cgcgtggctg gcacagggga gggtggcctg gaggagatgg tggaggagct caacagcggg 180 aaggtgatgt acgccttctg cagagtgaag gaccccaact ctggactgcc caaatttgtc 290 ctcatcaact ggacaggcga gggcgtgaac gatgtgcgga agggagcctg tgccagccac 300 gtcagcacca tggccagctt cctgaagggg gcccatgtga ccatcaacgc acgggccgag 360 gaggatgtgg agcctgagtg catcatggag aaggtggcca aggcttcagg tgccaactac 920 agctttcaca aggagagtgg ccgcttccag gacgtgggac cccaqgcccc agtgggctct 480 gtgtaccaga agaccaatgc cgtgtctgag attaaaaggg ttggtaaaga cagcttctgg 590 gccaaagcag agaaggagga ggagaaccgt cggctggagg aaaaqcggcg ggccqaggag 600 gcacagcggc agctggagca ggagcgccgg gagcgtgagc tgcgtgaggc tgcacgccgg 660 gagcagcgct atcaggagca ggqtggcgag qccagccccc agaggacgtg ggagcaqcag 720 caagaagtgg tttcaaggaa ccgaaatgag caggagtctg ccgtgcaccc gagggagatt 780 ttcaagcaga aggagagggc catgtccacc acctccatct ccagtcctca gcctggcaag 840 ctgaggagcc ccttcctgca gaagcagctc acccaaccag agacccactt tggcagagag 900 ccagctgctg ccatctcaag gcccagggca gatctccctg ctgaggagcc ggcgcccagc 960 actcctccat gtctggtgca ggcagaagag gaggctgtgt atgaggaacc tccagagcag 1020 gagaccttct acgagcagcc cccactggtg cagcagcaag gtgccggctc tgagcacatt 1080 gaccaccaca ttcagggcca ggggctcagt gggcaagggc tctgtgcccg tgccctgtac 1140 gactaccagg cagccgacga cacagagatc tcctttgacc ccgagaacct catcacgggd 1200 atcgaggtga tcgacgaagg ctgqtggcgt qgctatgggc cggatggcca ttttggcatg 1260 ttccctgcca actacgtgga gctcattgag tgaggctgag ggcggccgct agactagtct 1320 agagaaaaaa c 1331 <210> 9 <211> 760 <212> PRT
<213> Homo sapiens <220>
<221> UNSURE
<222> (16) <220>
<221> UNSURE
<222> (41) <220>
<221> UNSURE
<222> (99) <220>
<221> UNSURE
<222> (48) <220>
r4J ><~c' trA 7 .:.in Printed X20 p°g ~", ~. ~" ~
~2001-.~ 1 ~~kf°~ f ~G4~wS4h''~~ 2c4 -.___....._...__- '.~ X35.1:.:' ~..'CA 02375498 2001-12-17 <221> UNSURE
<222> (69) <220>
<221> UNSURE
<222> (96) <220>
<221> UNSURE
<222> 11121 <22:0>
<221> UNSURE
<222> (118) <220>
<221> UNSURE
<222> 4131) <220>
<221> UNSURE
<222> (199) <220>
<221> UNSURE
<222> (160) <220>
<221> UNSURE
<222> (168) <220>
<221> UNSORE
<222> (173) <220>
<221> UNSURE
<222> (192) <220>
<221> UNSURE
<222> (198) <220>
<221> UNSURE
<222> (208) <220>
B
p .
S.W . ~ ~'r~ n't ,Waw.., T =;t.

<221> UNSURE
<222> (227) <220>
<221> UNSURE
<222> (240) <220>
<221> UNSURE
<222> (256) <220>
<221> UNSURE
<222> (264) <220>
<221> UNSURE
<222> (276) <220>
<221> UNSURE
<222> (288) -<220>
<221> UNSURE
<222> (302) <220>
<221> UNSURE
<222> (309) <220>
<221> UNSURE
<222> (336) <220>
<221> UNSURE
<222> (352) <220>
<221> UNSURE
<222> (383) <220>
<221> UNSURE
<222> (3991 <220>

~~ "5y ~ ' w~t'~~' x E a,' : ~.
~'rinited:2~-09=2001 ' k ~ r ) > ~a~ _ ... ._. ,...,.. , .... a~,.slu_..,a~*,two.. e.,.d YSr ~...ae.:f~di.hi3.kY,~".a. _ "' g y ~~~a,~x~ -~-~a: ,.-~_... I , .. , . .. .

c <221> UNSURE
<222> (431) <220>
<221> UNSURE
<222> (947) <220>
<221> UNSURE
<222> (9~9) <220>
<221> UNSURE
<222> (995) <220>
<221> UNSURE
<222> (520) <220>
<221> UNSURE
<222> (527) <220>
<221> UNSURE
<222> (593) <220>
<221> UNSURE
<222> ;552) <220>
<221> UNSURE
<222> (575) <220>
<221> UNSURE
<222> (591) <220>
<221> UNSURE
<222> ffi22) <220>
<221> UNSURE
<222> (639) <220>
Y x s 1~' ~~; ~~v <: nx :.. :.<. ~ . . ~< ~~,cy'C. k ~F~.s<'sr~
L
,., ,.... . . . ... . . . . .. _< . ..W. "._ ~. ! ~.. .. '=.tari",:.

<221> UNSURE
<222> (671) <220>
<221> UNSURE
<222> (687) <220>
<221> UNSURE
<222> (719) <220>
<221> UNSURE
<222> (735 <900> 9 His Glu Ile Pro Thr Val Pro Thr Tyr Tyr Pro Ala Lys Pro Gln Xaa Glu Arg Ala Trp Arg Asn Gln Arg Gly Lys Lys Thr Leu Leu Ser Thr Leu Val Trp His Gly Glu Glu Thr Xaa Glu Val Xaa Asn Lys Trp Xaa Ala Pro Gly Ala Pro Pro Val Ser Pro Arg G1y Ala Arg Gly Gly Xaa Arg Pro Cys Gly Pro.Pro Val Lys Tyr His Tyr Ser Asp Arg Phe Thr Asp Pro Val Arg Arg Gly Gly Glu Pro Arg Gly Ala Leu Ala Ser Xaa g5 90 95 Ala Lys Arg Pro Ala Ala Arg Arg Pro Gly Ala Thr Arg Ser Gly Xaa Ala Arg Trp Gly Val Xaa Leu Gly Arg Tyr Thr Cys G1n Thr Val Gln Val Ser Xaa Gly Glu Leu Arg Glu Asp Arg Asn Leu Pro Trp Ser Xaa Arg Ala Lys Ala Arg Leu Ile Leu Ile Phe Ser Thr Asn Thr Asp Xaa Ser Gly Ala Ser Arg Ser Phe Xaa Pro Phe Gly Phe Xaa Ala Gly Val 165 170 17s r(tlt~ ~~ ~9=~C~O~ ' ~f? ~ ~~' ,:~ p C 't1 ~1=' ~ , yTS&'e.
.~.,s,1_., .. ,~, .... . a es ." ,....". ~hY~w'i4rf~~.rn.~.W Mi:"

.'~'yiwh,y~.~°'" . . . ~~..~I~~. 1:F,p.:. ...>.~'.,.:.

Arg Lys Val Thr Thr Gly Ile Thr Gly Leu Trp Arg Pro Ser Val Xaa Ser Asp Val Ala Phe Xaa Ser Phe Asp Val Gly Ser Ser Tyr His'Xaa Ala Glu Phe Thr Lys Arg Trp Ile Val His Pro Leu Ile Gly Asn Ser Trp Asp Xaa Thr Val Val Arg Gln Val Ser Phe Thr Leu Leu Met Xaa Cys Cys Cys His Gly Asn Pro Ala Gln Tyr Glu Arg Asn Arg Arg Xaa His Leu Val Tyr Val Leu Gly Xaa Gly Ala Asn Gly Ala Lys Leu Ser Val Gly Leu Xaa Leu Asn Ala Ser Lys Ser Glu Ser Arg Prfl Gly Xaa Thr Ile Arg Gln Arg Arg Gly Ala Ser Val Gly Leu Gly Xaa Pro Xaa Arg Leu Ser Pro Pro Ala Gly Arg Pro Pro Leu His Ala Pro Arg Arg Gly Arg Ala Arg Ala Pro Pro Arg Ala Gly Thr Gly Val Arg Cys Xaa Val Pro Phe Val Leu Gly Asn Gly Ala Arg Pro Glu Arg Arg Pro Xaa Arg Pro Ser Arg Thr Ala Arg Ser Trp Gly Thr Trp Arg Thr Ser Ile Ser Ser Pro Gln Pro Gly Lys Leu Arg Ser Pro Phe Leu Gln Xaa Gln Leu Thr Gln Pro Glu Thr His Phe Gly Arg Glu Pro Ala Ala Xaa Ser Arg Pro Arg Ala Asp Leu Pro Ala Glu Glu Pro Ala Pro Ser Pro Pro Cys Leu Val Gln Ala Glu Glu Glu Ala Val Tyr Glu Glu Pro Xaa Glu 420 425 930 j ~'~i~'l~f.'f.~#~~ ~~-~~~~~ ~
'a~ _. ....... . . _ ~~v'~:. ~'.at:~ar~ia~r.~ ~';S;IaCa'.v.-'.~. :,~'.
i Gln Glu Thr Phe Tyr Glu Gln Pro Pro Leu Val Gln Glt~ Gln Xaa Gly Ser Glu His Ile Asp His His Ile Gln Gly Gln Gly Leu Ser Gln Gly Leu Cys Ala Arg Ala Leu Tyr Asp Tyr Gln Ala Ala Asp Asp Xaa Glu Ile Ser Phe Asp Pro Glu Asn Leu Ile Thr Gly Ile Glu Val Xaa Glu 485 _ 990 495 Gly Trp Trp Arg Gly Tyr Gly Pro Asp Gly His Phe Gly Met Pro Ala Asn Tyr Val Glu Leu Ile Glu Xaa Gly Xaa Gly His Ile Leu Xaa Phe Pro Ser Gln Thr Trp Leu Pro Tyr Cys Trp Lys Arg Arg Pro Xaa Xaa His Ser Ala Leu Phe Gln Glu Xaa Asp Pro Gln Xaa Gly Xaa Leu Arg Ala Pro Ser Gly Leu Ala Asp Ser Ala Cys His Pro Lys Cys Xaa Asn Gly Leu Val Ile Pro Thr His Pro Ser Cys Ile Pro Arg Pro Xaa Thr Ala Trp Leu Leu Pro Leu Thr Gly Tyr Xaa Ala Lys Pro Cys Trp Pro -Ser Pro Glu Trp Pro Leu Pro Ser Cys Gly Glu Gly Ser Xaa Xaa Gly Ala Ser Gly Arg Leu Trp Leu Pro Ser Ala Phe Ile Cys Leu Xaa Phe Ser Leu Ala Ser Lys Gly Trp Trp Pro Pro Leu Phe Arg Met Leu Gly Asn Ser Glu Arg Arg Glu Leu Phe Leu Ala Glu Phe Val Thr Xaa Val Arg Val Asp His Gly Gly Leu Ala Ala Gly Asn Leu Ser Cys Xaa Leu ~'tn~eda00~ 20~~ ~ '' i'~ , , a , .,.
1 ~~ . ~ ~ j H
w ..Vwusn:~;~~" s..~wiv'oxa~~~YC.~
-a..S~_,.,lM,ur.u,~ »..,a., ws..a4vZ.3vuu-..~w,-Leu Cys Ala Pro His Ser Ile Ser Leu Ser Leu Cys Leu Gly Gly Lys Trp Gly Cys Arg Trp Pro Ser Ser His Pro Gly Tyr Ser Lys Xaa Ala Asp Thr Thr Cys Ser Ser Thr Arg Leu Thr Arg Cys Leu Gin Xaa Val Cys Ala Ser Thr Asp Ser Asp Phe Arg Lys Ser Lys Lys Lys Lys Lys Leu Glu Lys Leu Trp Thr Ser Ser <210> 10 <211> 753 <212> PRT

<213> Homo Sapiens <220>
<221> UNSURE
<222> (10) <220>
<221> UNSURE
<222> (13) <220>
<221> UNSURE
<222> (32) <220>
<221> UNSURE
<222> (98) <220>
<221> UNSURE
<222> (80) <220>
<221> UNSURE
<222> (87) <220>
<221> UNSURE

~rinx~d~~~~-09 ,:200'1 r _ . . ....", . ,.~~ , __, ...,~, t ,,>.,t~;.~~ ~ ~'.~

<222> (96) <220>
<221> UNSURE
<222> (100) <220>
<221> UNSURE
<222> (127) <220>
<221> UNSURE
<222> (137) <220>
<221> UNSURE
<222> (142) <220>
<221> UNSURE
<222> (194) <220>
<221> UNSURE
<222> (166) <220>
<221> UNSURE
<222> (176) <220>
<221> UNSURE
<222> (192) <220>
<221> UNSURE
<222> (196) <220>
<221> UNSURE
<222> (224) <220>
<221> UNSURE
<222> (233) <220>
<221> UNSURE
-.
Pi~tnfd:20 Og-2Q01 ' y~p~ l~ ~ ~,~ ~ ~'~' . . . .
~ , , ~,.

<222> (240) <220>
<221> UNSURE
<222> (245) <220>
<221> UNSURE
<222> (272) <220>
<221> UNSURE
<222> (288) <220>
<221> UNSURE
<222> (320) <220>
<221> UNSURE
<222> (336) <220>
<221> UNSURE
<222> (352)..(353) <220>
<221> UNSURE
<222> (363) <220>
<221> UNSURE
<222> (368) <220>
<221> UNSURE
<222> (371) <220>
<221> UNSURE
<222> (383)..(385) <220>
<221> UNSURE
<222> (410) <220>
<221> UNSURE

~R~~I'I~~~I ~~I'-~0~,-~~~~"I' . .~,.,~ . _w,... , x . .... . .,_ ,.. ,.. . .~~~ ..~ . ~.~,~.-=o"°.k,_ h. _~'1 ... .., , ._ .

<222> (916) <220>
<221> UNSURE
<222> (432) <220>
<221> UNSURE
<222> (438) <220>
<221> UNSURE
<222> (964) <220>
<221> UNSURE
<222> (480) <220>
<221> UNSURE
<222> (487) <220>
<221> UNSURE
<222> (495)..(997) <220>
<221> UNSURE
<222> (505) <220>
<221> UNSURE
<222> (512)..(513) <220>
<221> UNSURE
<222> (522) <220>
<221> UNSURE
<222> (528) <220>
<221> UNSURE
<222> (550) <220>
<221> UNSURE
r.~* , .
P~riii~~~~0~'a8~t301:~ ~ ~~ ~:~~ '~ ~ n=:. . .
.. a ..... ..........:r:. a ."x..~.~.~... ~R~._ ,~:'e~'~u'.~z°f"" -'~~''s'L'"-'°"":~''~"'"::'.~r""'.

~~~~fa~~ ~iy~~~~o 1 t .

<222> (554) <220>
<221> UNSURE
<222> (560) <220>
<221> UNSURE
<222> (579) <220>
<221> UNSURE
<222> (576) <220>
<221> UNSURE
<222> (589) <220>
<221> UNSURE
<222> (591) <220>
<221> UNSURE
<222> (608) <220>
<221> UNSURE
<222> (624) <220>
<221> UNSURE
<222> (652) <220>
<221> UNSURE
<222> 1656)..(657) <220>
<221> UNSURE
<222> 1662)..(663) <220>
<221> UNSURE
<222> 1672) <220>
<221> UNSURE
,~"" _:~
r , ~lntecl.~0 t~9. ~~001' ~~ . , _ a _ . _...._. . ~ s .. _~: ~ _ .._ ~~ P

<222> (702) <220>
<221> UNSURE
<222> (704) <220>
<221> UNSURE
<222> (718) <220>
<221> UNSURE
<222> ('120) <220>
<221> UNSURE
<222> (723) <220>
<221> UNSURE
<222> (734) <220>
<221> UNSURE
<222> (743) <220>
<221> UNSURE
<222> (752) <400> 10 Ile Arg His Glu His Gly Glu Glu Thr Xaa Glu Val Xaa Asn Lys Glu Ala Pro Gly Ala Pro Pro Val Ser Pro Arg Gly Ala Arg Gly Gly Xaa Arg Arg Pro Cys Gly Pro Pro Val Lys Tyr His Tyr Ser Asp Arg Xaa Thz Asp Pro Val Arg Arg Gly Gly Glu Pro Arg Gly Ala Leu Ala Gly Ala Lys Arg Pro Ala Ala Arg Arg Pro Gly Ala Thr Arg Ser Gly Xaa Ser Ala Arg Trp Gly Val Xaa Leu Gly Arg Tyr Thr Cys Gln Thr Xaa ~~i~a~ rrW,s'~"~~r~ir~~- . ~s.
P~rrrtted 20 09-2fl01 i epo ) .. . . ... _.., _. ~ . _ . _,. .. -Gln Val 5er Xaa Gly Glu Leu Arg Glu Asp Arg Asn Leu Pro Trp Arg Arg Ala Lys Ala Arg Leu Ile Leu Ile Phe Ser Thr Asn Thr Asp Xaa Glu Ser Gly Ala Ser Arg Ser Phe Xaa Pro Phe Gly Phe Xaa Ala Xaa Val Arg Lys Val Thr Thr Gly Ile Thr Gly Leu Trp Arg Pro Ser His Ser Asp Val Ala Phe Xaa Ser Phe Asp Val Gly Ser Ser Tyr His Xaa Glu Ala Glu Phe Thr Lys Arg Trp Ile Val His Pro Leu Ile Gly Xaa Ser Trp Val Xaa Thr val Val Arg Gln Val Ser Phe Thr Leu Leu Met Cys Cys Cys His Gly Asn Pro Ala Gln Tyr Glu Arg Asn Arg Arg Xaa Arg His Leu Val Tyr Val Leu Gly Xaa Gly Ala Asn Gly Ala Lys Xaa Ser Val Gly Leu Xaa Leu Asn Ala Ser Lys Ser Glu Ser Arg Pro Gly Thr Ile Arg Gln Arg Arg Gly Ala Ser Val Gly Leu Gly Xaa Pro Xaa Pro Arg Leu Ser Pro Pro Ala Gly Arg Pro Pro Pro Ser Thr Arg Xaa Arg Ala Gly Gly Arg Val Pro Arg Arg Ala Pro Gly Pro Gly Ser Ala Glu Cys Pro Ser Ser Trp Glu Thr Gly Arg Gly Arg Lys Gly Gly Xaa Pro Leu Ala Arg His Ala Pro His Val Arg Ala Arg Ala Glu Phe Xaa Ser Ser Thr Ile His Asn Arg His Thr Ser Ala Cys Ile Phe Met Xaa . Pr~ntsd.~D-09=2001 ~~p~Ii~e'~~ ,... ~ ~~rl~p~c~io~=

Xaa Ile Leu Phe Leu Trp Val Asp Ile Gln Xaa Trp Asp Cys Xaa Xaa Thr Trp Xaa Phe Tyr Phe Trp Phe Ile Glu Lys Ser Ser Tyr Xaa Xaa Xaa Arg Leu Tyr Lys Phe Thr Ser Leu Pro Ser Asp Phe Phe Lys Glu Arg Met Val Trp Arg Asn Ala Pro His Xaa Tyr Pro Pro Phe Thr Xaa Leu Leu Gln Asn Asp Phe Lys Gly Tyr Arg Tyr Leu Gln Val Ser Xaa Arg Gln Ile Glu Tyr Xaa Asn Phe Cys Ile Arg Gly Thr Asp Phe Ile Gln Ser Cys Met Asn Lys Asp Lys Cys Ser Arg Asp Leu Gln Ser Xaa Asn Trp Lys Ser Gln Met Lys Tyr Ile Ser Ser Ser Thr Thr Ser Xaa Ser Thr Glu Leu Ala Leu Xaa Ser Ser Leu Ile Pro Thr Tyr Xaa Xaa Xaa Lys Gly Phe Ile Ser Asn Iie Leu Xaa Gly Ile Lys Ile Lys Xaa Xaa Val Lys Leu Phe Ser Leu Ala Phe Xaa Phe Gln Asn Ile Lys Xaa Pro Ser Ile Gly His Leu Tyr Cys Thr Arg His Cys Val Cys His Ser Lys Met Phe Ser Trp Xaa Cys Ser Gln Xaa Phe Cys Arg Val Arg Xaa Ser Leu Thr Val Val Arg Leu Phe Ser Lys Arg Asn Leu Xaa Thr Xaa Phe Asn Leu Arg Lys Val Ser Asn Arg Thr Arg Thr Xaa Thr Xaa Ile Thr Leu Gln Ile Ser Pro Tyr His Thr Ala Ser Thr Cys Ala Cys Xaa ~ ~~ ~r~~~p~~r~~~ . s~~
R'~inted:20~-Q9=2001 '' ~ ~ rz.: .
.. ~x ~P_.,~. .~H~a~h.~. ~ ,~ ... ~.._ .. ..

_ ~ CA 02375498 2001-12-17 Leu Ile Pro Gly Ser Cys Tyr Phe Pro Phe Tyr Phe Leu-Ser Leu Xaa Thr Thr Pro Phe Ser Pro His Phe Phe Ser Phe Phe heu Ile Val Tyr Ile Thr Asn Thr Cys Leu Ser Glu Gln Leu Ile Xaa His Lys Arg Xaa Xaa Ser Thr Gly Glu Xaa Xaa Leu Ile Pro Val Ile Leu Ala Leu Xaa Ala Lys Ala Gly Arg Ser Leu Glu Ser Arg Val Arg Asp Gln Pro Gln His Gly Glu Thr Leu Ser Leu Gln Lys Asn Thr Lys Ile Xaa Pro Xaa Val Leu Ala His Thr Cys Ser Leu Ser Tyr Ser Glu Gly Xaa Gly Xaa Ile Asp Xaa Ala Gln Glu Val Glu Ala Ala Ala Val Arg Xaa Asp Ala Ile Ala Leu Gln Pro Gly Xaa Glu Arg Glu Thr Leu Ser Gln Lys Xaa Lys w ~'t~
P~rii~ted 20-~9-2~4~:~ ~~pc~r~~ ~='F~e~ras~es ~~~
x . _ . .. ... . . _ ~.. _. r_ _. .~ ~ ~,a p. ~ ,_..~.. _ _

Claims (11)

WE CLAIM:

1. A protein produced by an epithelial cell and having epithelial cell growth inhibitory properties the protein having a molecular weight of approximately kilodaltons.

2. The protein 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. The protein of claim 1, wherein the epithelial cells are colon, ovary, prostate, spleen, testis, or thymus cells.

4. The protein of claim 1, wherein the protein is encoded by a nucleic acid sequence having substantial identity over at least one third of a nucleic acid sequence selected form Seq Id Nos: 1, 2, 3, and 4.

5. The protein of claim 1, wherein the protein is specifically recognized by the anti-mammastatin antibody, 7G6.

6. A nucleic acid sequence comprising SEQ ID NOs: 1, 2, 3, 4, or a nucleic acid sequence having substantial identity over at least one third of the sequence with SEQ ID NOs: 1, 2, 3, or 4.

7. 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 6; and correlating a reduction or absence of said protein or said nucleic acid sequence with epithelial cell cancer.

8. 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 6.

9. A diagnostic kit comprising:
the protein of claim 1 or the nucleic acid sequence of claim 6; and optionally an antibody that specifically binds the protein of claim 1.

10. The diagnostic kit of claim 9, wherein the antibody is 7G6.

11. Use of the protein of claim 1 for he manufacture of medicament for the treatment of epithelial cell cancer.

39a