Nuclear matrix protein alterations associated with colon cancer and colon metastasis to the liver, and uses thereof

Abstract

Proteins useful in the diagnosis of proliferative disorders of the colon are present in nuclear matrix protein preparations and can be characterized by molecular weight, isoelectric point, and amino acid sequence. The proteins may be identified, for example, by 2D-gel electrophoresis or by specific binding partners, such as antibodies.

What is claimed is:  
     
         1 . A purified protein which is present in cancerous colon cells but absent or reduced in amount in normal colonic epithelial cells, said protein selected from the group consisting of: 
 a) CC2 having a molecular weight of about 56 kD and a pI of about 6.22;    b) CC3 having a molecular weight of about 43 kD and a pI of about 6.3;    c) CC4 having a molecular weight of about 43 kD and a pI of about 6.2;    d) CC5 having a molecular weight of about 42 kD and a pI of about 6.2;    e) CC6a having a molecular weight of about 20 kD and a pI of about 6.9;    f) CC6b having a molecular weight of about 20 kD and a pI of about 6.8.    g) L1 having a molecular weight of about 50 kD and a pI of about 6.01;    h) L2 having a molecular weight of about 20 kD and a pI of about 5.73; and    i) L5 having a molecular weight of about 19 kD and a pI of about 5.88.    
     
     
         2 . The purified protein of  claim 1  wherein said protein is CC2.  
     
     
         3 . The purified protein of  claim 1  wherein said protein is CC3.  
     
     
         4 . The purified protein of  claim 1  wherein said protein is CC4.  
     
     
         5 . The purified protein of  claim 1  wherein said protein is CC5.  
     
     
         6 . The purified protein of  claim 1  wherein said protein is CC6a.  
     
     
         7 . The purified protein of  claim 1  wherein said protein is CC6b.  
     
     
         8 . The purified protein of  claim 1  wherein said protein is L1.  
     
     
         9 . The purified protein of  claim 1  wherein said protein is L2.  
     
     
         10 . The purified protein of  claim 1  wherein said protein is L5.  
     
     
         11 . The purified protein of  claim 1  wherein said protein is more detectable in colon cancer metastasis to liver than in normal liver tissue.  
     
     
         12 . The purified protein of  claim 11  wherein said protein is L1, L2 or L3.  
     
     
         13 . The purified protein of  claim 2  wherein CC2 comprises an amino acid sequence shown as SEQ ID NO: 14, 15, 16, 17 or 23.  
     
     
         14 . The purified protein of  claim 3  wherein CC3 comprises an amino acid sequence shown as SEQ ID NO: 24-26.  
     
     
         15 . The purified protein of  claim 4  wherein CC4 comprises an amino acid sequence shown as SEQ ID NO: 27-29.  
     
     
         16 . The purified protein of  claim 5  wherein CC6a comprises an amino acid sequence shown as SEQ ID NO: 18-20.  
     
     
         17 . The purified protein of  claim 6  wherein CC6b comprises an amino acid sequence shown as SEQ ID NO: 21.  
     
     
         18 . The purified protein of  claim 9  wherein L2 comprises an amino acid sequence shown as SEQ ID NO: 30-32.  
     
     
         19 . The purified protein of  claim 10  wherein L5 comprises an amino acid sequence shown as SEQ ID NO: 33-36.  
     
     
         20 . The purified protein of  claim 1 , wherein said protein is present in a nuclear matrix preparation.  
     
     
         21 . A binding partner specific for the purified protein of  claim 1 .  
     
     
         22 . The purified protein of  claim 21 , wherein said binding partner is a monoclonal antibody.  
     
     
         23 . The purified protein of  claim 21 , wherein said binding partner is a polyclonal antibody.  
     
     
         24 . A purified protein which is present in normal epithelial colonic cells but absent or reduced in amount cancerous colon cells, said protein selected from the group consisting of: 
 a) N1 having a molecular weight of about 40 kD and a pI of about 5.5;    b) N2 having a molecular weight of about 30 kD and a pI of about 5.94;    c) N3 having a molecular weight of about 30 kD and a pI of about 5.88;    d) N4 having a molecular weight of about 30 kD and a pI of about 5.80;    e) N5 having a molecular weight of about 30 kD and a pI of about 5.73; and    f) N6 having a molecular weight of about 18 kD and a pI of about 6.6.    
     
     
         25 . The purified protein of  claim 24  wherein said protein is N1.  
     
     
         26 . The purified protein of  claim 24  wherein said protein is N2.  
     
     
         27 . The purified protein of  claim 24  wherein said protein is N3.  
     
     
         28 . The purified protein of  claim 24  wherein said protein is N4.  
     
     
         29 . The purified protein of  claim 24  wherein said protein is N5.  
     
     
         30 . The purified protein of  claim 24  wherein said protein is N5.  
     
     
         31 . The purified protein of  claim 24 , wherein said protein is present in a nuclear matrix preparation.  
     
     
         32 . A binding partner specific for the purified protein of  claim 24 .  
     
     
         33 . The purified protein of  claim 32 , wherein said binding partner is a monoclonal antibody.  
     
     
         34 . The purified protein of  claim 32 , wherein said binding partner is a polyclonal antibody.  
     
     
         35 . A purified protein which is present in primary colon cancer cells and liver metastasis derived therefrom and is useful as a cancer diagnostic marker, said protein selected from the group consisting of: 
 a) L3 having a molecular weight of about 18 kD and a pI of about 6.09; and    b) L4 having a molecular weight of about 18 kD and a pI of about 6.00.    
     
     
         36 . The purified protein of  claim 35 , wherein said protein is L3.  
     
     
         37 . The purified protein of  claim 35 , wherein said protein is L4.  
     
     
         38 . The purified protein of  claim 35 , wherein said protein is present in a nuclear matrix preparation.  
     
     
         39 . A binding partner specific for the purified protein of  claim 35 .  
     
     
         40 . The purified protein of  claim 39  wherein said binding partner is a monoclonal antibody.  
     
     
         41 . The purified protein of  claim 39 , wherein said binding partner is a polyclonal antibody.  
     
     
         42 . A method of producing polyclonal antibodies that differentiate between cancerous and normal colonic epithelial cells comprising immunizing an animal with the protein of  claim 1  and recovering said antibodies.  
     
     
         43 . A method of producing polyclonal antibodies that differentiate between cancerous and normal colonic epithelial cells comprising immunizing an animal with the protein of  claim 24  and recovering said antibodies.  
     
     
         44 . A method of producing polyclonal antibodies that differentiate between colon cancer metastases to liver and normal liver epithelial cells comprising immunizing an animal with the protein of  claim 35  and recovering said antibodies.  
     
     
         45 . A method~of producing a monoclonal antibody that differentiates between cancerous and normal colonic epithelial cells comprising immunizing an animal with the protein of  claim 1 , removing B cells from the immunized animal and: i) immortalizing and isolating said B cells that produce an antibody specific for said protein or ii) obtaining nucleic acid encoding antibody heavy and light chains from the cells, transforming, transfecting or transducing host cells with one or more expression vectors containing the nucleic acid such that the host cells produce antibody encoded by said nucleic acid, and selecting host cells produce an antibody specific for the protein.  
     
     
         46 . A method of producing a monoclonal antibody that differentiates between cancerous and normal colonic epithelial cells comprising immunizing an animal with the protein of  claim 24 , removing B cells from the immunized animal and: i) immortalizing and isolating said B cells that produce an antibody specific for said protein or ii) obtaining nucleic acid encoding antibody heavy and light chains from the cells, transforming, transfecting or transducing host cells with one or more expression vectors containing the nucleic acid such that the host cells produce antibody encoded by said nucleic acid, and selecting host cells produce an antibody specific for the protein.  
     
     
         47 . A method of producing a monoclonal antibody that differentiates between colon cancer metastases to liver and normal liver epithelial cells comprising immunizing an animal with the protein of  claim 35 , removing B cells from the immunized animal and: i) immortalizing and isolating said B cells that produce an antibody specific for said protein or ii) obtaining nucleic acid encoding antibody heavy and light chains from the cells, transforming, transfecting or transducing host cells with one or more expression vectors containing the nucleic acid such that the host cells produce antibody encoded by said nucleic acid, and selecting host cells produce an antibody specific for the protein.  
     
     
         48 . A method of diagnosing a proliferative disorder of the colon in a patient, said method comprising: analyzing tissue, stool or body fluid from the patient for the presence of at least one protein that is present in cancerous colonic cells but absent or reduced in amount in normal colonic epithelial cells, or present in normal colonic epithelial cells but absent or reduced in amount in cancerous colonic cells, wherein said protein is selected from the group consisting of: 
 a) CC2 having a molecular weight of about 56 kD and a pI of about 6.22;    b) CC3 having a molecular weight of about 43 kD and a pI of about 6.3;    c) CC4 having a molecular weight of about 43 kD and a pI of about 6.2;    d) CC5 having a molecular weight of about 42 kD and a pI of about 6.2;    e) CC6a having a molecular weight of about 20 kD and a pI of about 6.9;    f) CC6b having a molecular weight of about 20 kD and a pI of about 6.8.    g) L1 having a molecular weight of about 50 kD and a pI of about 6.01;    h) L2 having a molecular weight of about 20 kD and a pI of about 5.73;    i) L5 having a molecular weight of about 19 kD and a pI of about 5.88;    j) N1 having a molecular weight of about 40 kD and a pI of about 5.5;    k) N2 having a molecular weight of about 30 kD and a pI of about 5.94;    l) N3 having a molecular weight of about 30 kD and a pI of about 5.88;    m) N4 having a molecular weight of about 30 kD and a pI of about 5.80;    n) N5 having a molecular weight of about 30 kD and a pI of about 5.73; and    o) N6 having a molecular weight of about 18 kD and a pI of about 6.6.    
     
     
         49 . The method of  claim 48  wherein the presence of said at least one protein is detected by 2 dimensional gel electrophoresis.  
     
     
         50 . The method of  claim 48  wherein the presence of said at least one protein is detected with a binding partner specific for the protein.  
     
     
         51 . The method of  claim 50  wherein said binding partner is a monoclonal or polyclonal antibody.  
     
     
         52 . The method of  claim 48  wherein the presence of said at least one protein is determined by detecting mRNA encoding the protein through hybridization with a nucleic acid.  
     
     
         53 . The method of  claim 48 , wherein said purified protein is present in a nuclear matrix preparation.  
     
     
         54 . A method of diagnosing colon cancer in a patient, said method comprising: analyzing tissue, stool or body fluid from the patient for the presence of calreticulin.  
     
     
         55 . The method of  claim 54  wherein said detection is by two-dimensional gel electrophoresis.  
     
     
         56 . The method of  claim 54  wherein calreticulin is detected by a specific binding partner.  
     
     
         57 . The method of  claim 56  wherein the specific binding partner is a monoclonal or polyclonal antibody.  
     
     
         58 . The method of  claim 56  wherein calreticulin is detected by hybridizing with a nucleic acid for calreticulin.  
     
     
         59 . The method of  claim 54  wherein tissue is analyzed.  
     
     
         60 . The method of  claim 59  where said tissue is a nuclear matrix preparation.  
     
     
         61 . The method of  claim 54  wherein said proliferative disorder is colorectal carcinoma.  
     
     
         62 . The method of  claim 54  wherein said proliferative disorder is colonic adenoma.  
     
     
         63 . The method of  claim 62  wherein said proteins are CC3, CC4 or CC5.  
     
     
         64 . A method of evaluating colonic adenomas for potential to become malignant, said method comprising: analyzing the adenoma for the presence of at least one protein that is present in cancerous colonic cells but absent or reduced in amount in normal colonic epithelial cells, or present in normal colonic epithelial cells but absent or reduced in amount in cancerous colonic cells, wherein said protein is selected from the group consisting of: 
 a) CC2 having a molecular weight of about 56 kD and a pI of about 6.22;    b) CC3 having a molecular weight of about 43 kD and a pI of about 6.3;    c) CC4 having a molecular weight of about 43 kD and a pI of about 6.2;    d) CC5 having a molecular weight of about 42 kD and a pI of about 6.2;    e) CC6a having a molecular weight of about 20 kD and a pI of about 6.9;    f) CC6b having a molecular weight of about 20 kD and a pI of about 6.8.    g) L1 having a molecular weight of about 50 kD and a pI of about 6.01;    h) L2 having a molecular weight of about 20 kD and a pI of about 5.73;    i) L5 having a molecular weight of about 19 kD and a pI of about 5.88;    j) N1 having a molecular weight of about 40 kD and a pI of about 5.5;    k) N2 having a molecular weight of about 30 kD and a pI of about 5.94;    l) N3 having a molecular weight of about, 30 kD and a pI of about 5.88;    m) N4 having a molecular weight of about 30 kD and a pI of about 5.80;    n) N5 having a molecular weight of about 30 kD and a pI of about 5.73; and    o) N6 having a molecular weight of about 18 kD and a pI of about 6.6; and    wherein detection of said proteins indicates an increased potential to become malignant.    
     
     
         65 . The method of  claim 64  wherein the presence of said at least one protein is detected by 2 dimensional gel electrophoresis.  
     
     
         66 . The method of  claim 64  wherein the presence of said at least one protein is detected with a binding partner specific for the protein.  
     
     
         67 . The method of  claim 66  wherein said binding partner is a monoclonal or polyclonal antibody.  
     
     
         68 . The method of  claim 64  wherein the presence of said at least one protein is determined by detecting mRNA encoding the protein through hybridization with a nucleic acid.  
     
     
         69 . The method of  claim 64  wherein said proteins are CC3, CC4 and CC5.  
     
     
         70 . A method of diagnosing colon cancer to liver metastasis in a patient, said method comprising: analyzing a sample of liver for the presence of at least one protein, wherein said protein is selected from the group consisting of: 
 a) L1 having a molecular weight of about 50 kD and a pI of about 6.01;    b) L2 having a molecular weight of about 20 kD and a pI of about 5.73;    c) L3 having a molecular weight of about 17 kD and a pI of about 6.09;    d) L4 having a molecular weight of about 17 kD and a pI of about 6.00; and    e) L5 having a molecular weight of about 19 kD and a pI of about 5.88.    
     
     
         71 . The method of  claim 70 , wherein the presence of said at least one protein is detected by high resolution, two-dimensional gel electrophoresis.  
     
     
         72 . The method of  claim 70 , wherein the presence of said at least one protein is detected with a binding partner specific for the protein.  
     
     
         73 . The method of  claim 72 , wherein said binding partner is a monoclonal or polyclonal antibody.  
     
     
         74 . The method of  claim 72 , wherein said protein is detected by hybridizing with a nucleic acid for calreticulin.  
     
     
         75 . A diagnostic assay kit for determining the presence of the protein of  claim 1 , comprising at least one binding partner which is capable of specifically binding with the protein.  
     
     
         76 . The diagnostic assay kit of  claim 75 , wherein said binding partner is immobilized on a solid support.  
     
     
         77 . The diagnostic assay kit of  claim 75  wherein said binding partner is detectably labeled.  
     
     
         78 . The diagnostic assay kit of  claim 75 , wherein said binding partner is an antibody.