Human cyclooxygenase-2 cDNA and assays for evaluating cyclooxygenase-2 activity

Abstract

The invention discloses a human cyclooxygenase-2 cDNA, a human cyclooxygenase-2 and assays for preferentially and independently measuring cyclogenase-2 or cyclooxygenase-1 activity present in a given sample.

1. An assay for determining the cyclooxygenase-2 activity of a sample  of a composition comprising a human osteosarcoma cell preparation, a sample and arachidonic acid, the assay comprising the steps of:
 (a) preparing the composition adding 
 (1) a  the human osteosarcoma cell preparation,  
 (2) a  the sample, said sample comprising a putative cyclooxygenase-2 inhibitor, and  
 (3) the arachidonic acid; and  
 
 (b) determining the amount of prostaglandin E 2  produced in step (a).  
 
     
     
       2. An assay for determining the cyclooxygenase-2 activity of a sample  according to  claim 1  comprising the steps of:
 (a) adding 
 (1) a human osteosarcoma cell preparation,  
 (2) a sample, said sample comprising a putative cyclooxygenase-2 inhibitor, and  
 (3) arachidonic acid; and  
 
 (b) determining the amount of prostaglandin E 2  produced in step (a),  
 wherein the cell preparation comprises 10 3  to 10 9  whole cells of osteosarcoma per cc, or 50 to 500 ug of osteosarcoma microsomes per ml of preparation; and 0.1 to 50 μl of arachidonic acid per ml of preparation.  
 
     
     
       3. An assay for determining the cyclooxygenase-2 activity of a sample a composition comprising a human osteosarcoma cell preparation, a sample and arachidonic acid, the assay comprising the steps of:
 (a) preparing the composition by adding 
 (1) a  the human osteosarcoma cell preparation,  
 (2) a  the sample, said sample comprising a putative cyclooxygenase-2 inhibitor, and  
 (3) the arachidonic acid; and  
 
 (b) determining the amount of prostaglandin E 2  produced in step (a)  
 (c) corrolating  correlating the amount of prostaglandin E 2  produced with cyclooxygenase-2 activity,  
 wherein the osteosarcoma cell preparation consists essentially of osteosarcoma 143.98.2 microsomes.  
 
     
     
       4. An assay according to  claim 3  wherein the osteosarcoma 143.98.2 microsomes are substantially free of endogenous arachidonic acid. 
     
     
       5. An assay according to  claim 3  wherein the microsomes are contacted with an amount of delipidized serum protein effective to reduce the amount of endogenous arachidonic acid in the microsomes by a factor of at least approximately 2. 
     
     
       6. An assay for determining the cyclooxygenase-2 activity of a sample  a composition comprising a human osteosarcoma cell preparation, a sample and arachidonic acid, the assay comprising the steps of:
 (a) preparing the composition by adding 
 (1) a  the human osteosarcoma cell preparation,  
 (2) a  the sample, said sample comprising a putative cyclooxygenase-2 inhibitor, and  
 (3) the arachidonic acid; and  
 
 (b) determining the amount of prostaglandin E 2  produced in step (a),  
 (c) corrolating  correlating the amount of prostaglandin E 2  produced with cyclooxygenase-2 activity,  
 wherein the human osteosarcoma cell preparation contains no recombinant vector.  
 
     
     
       7. An assay for determining the cyclooxygenase-2 activity of a sample  a composition comprising a human osteosarcoma cell preparation, a sample and arachidonic acid, the assay comprising the steps of:
 (a) preparing the composition by adding 
 (1) a  the human osteosarcoma cell preparation,  
 (2) a  the sample, said sample comprising a putative cyclooxygenase-2 inhibitor, and  
 (3) the arachidonic acid; and  
 
 (b) determining the amount of prostaglandin E 2  produced in step (a)  
 (c) corrolating  correlating the amount of prostaglandin E 2  produced with cyclooxygenase-2 activity,  
 wherein the osteosarcoma cell preparation consists essentially of whole cells of osteosarcoma 143.98.2.  
 
     
     
       8. A composition comprising:
 (a) an osteosarcoma cell preparation, having 10 3  to 10 9  osteosarcoma cells per cc of cell preparation or 50 to 500 μg of osteosarcoma microsomes; and    (b) 0.1 to 50 μl of arachidonic acid per cc of cell preparation.    
     
     
       9. A composition according to  claim 8  comprising 8×10 4  to 2×10 6  osteosarcoma 143.98.2 whole cells per cc of cell preparation or 100 to 400 μg of osteosarcoma 143.98.2 microsomes; and 10 to 20 μl of peroxide-free arachidonic acid per cc of cell preparation. 
     
     
       10. A composition according to  claim 9  wherein the microsomes are substantially free of endogenous arachidonic acid. 
     
     
       11. An assay for determining the cyclooxygenase-1 activity of a sample  a composition comprising the steps of:
 (a) preparing the composition by adding 
 (1) a COX-1 cell preparation, wherein the cell preparation consists essentially of whole cells of U-   937  or U -   937  microsomes,   
 (2) a sample, said sample comprising a putative cyclooxygenase-1 inhibitor;  
 (3) arachidonic acid; and  
 
 (b) determining the amount of prostaglandin E 2  produced in step (a)  
 (c) corrolating the amount of prostaglandin E 2  produced with cyclooxygenase-2 activity .  
 
     
     
       12. An assay according to  claim 11  wherein the COX-1 cell preparation consists essentially of whole cells of U-937. 
     
     
       13. An assay according to  claim 11  wherein the COX-1 cell preparation consists essentially of U-937 microsomes. 
     
     
       14. An assay according for  claim 11  for determining the cyclooxygenase-1 activity of a sample according to  claim 10  comprising the steps of:
 (a) adding  
 (1) a COX-1 cell preparation, 
 (2) a sample, said sample comprising a putative cyclooxygenase-1 inhibitor;  
 (3) arachidonic acid; and  
 
 (b) determining the amount of prostaglandin E 2  produced in step (a),  
 wherein the cell preparation comprises 10 5  to 10 8  whole cells of U-937 per cc, or 1 to 10 mg of U-937 microsomes per ml of preparation; and  
 0.1 to 50 μl of arachidonic acid per ml of preparation.  
 
     
     
       15. An assay according to  claim 14  wherein the cell preparation comprises 8×10 8  to 1.5×10 6  whole cells of U-937 per cc, or 1 to 5 mg of U-937 microsomes per ml of preparation. 
     
     
       16. Human Cyclooxygenase-2 cDNA which encodes protein of SEQ ID NO:10. 
     
     
       17. Human Cyclooxygenase-2 cDNA according to  claim 15  comprising the coding region which is bases 97 to 1909 of  FIG. 2  (SEQ ID NO: 11:).    
     
     
       18. Human Cyclooxygenase-2 which is shown in  FIG. 1  (SEQ ID NO: 10:).    
     
     
       19. An isolated A transformed  host cell that expresses cyclooxygenase-2 as shown in  FIG. 1  (SEQ. ID. NO. 10:) comprising:  wherein said host cell is transformed with 
 (a)  a mammalian or eukaryotic expression vector; and  
 (b)  comprising a sequence encoding human  said cyclooxygenase-2 comprising bases 97 to 1909 as shown in  FIG. 2  (  of SEQ ID NO:11)  or encodes protein of  FIG. 1  (  the polypeptide SEQ ID NO: 10) .    
 
     
     
       20. A system according to  claim 19  wherein the expression vector is a vacinia or baculovirus vector. 
     
     
       21. A system according to  claim 19  wherein the cyclooxygenase-2 is expressed in COS-7 cells. 
     
     
       22. An isolated cyclooxygenase-   2  cDNA comprising the coding region which is bases  97  to  1909  of SEO. ID. NO:  11 .   
     
     
       23. A recombinant cyclooxygenase-   2  consisting of SEO. ID. NO:  10 .   
     
     
       24. An isolated cyclooxygenase-   2  consisting of SEO. ID. NO:  10 .   
     
     
       25. A purified cyclooxygenase-   2  consisting of SEO. ID. NO:  10 .   
     
     
       26. The transformed host cell according to  claim 19  wherein the expression vector is a vacinia or baculovirus vector. 
     
     
       27. The transformed host cells according to  claim 19  wherein said host cells are COS-   7  cells.