Rapid methods for identifying modifiers of cellular apoptosis activity

Abstract

The invention provides a single-well, microscale method of determining the specific apoptotic activity of a cell. The method consists of contacting a cell population of about 1×10 5 cells for a time period of between about 30 minutes and 4 hours with a sufficient volume of medium containing an apoptotic specific diagnostic reagent and a diagnostic accessory reagent so as to cover the cell population, and determining the activity of the apoptotic specific diagnostic reagent. The invention also provides a method of identifying a compound which induces apoptosis. The invention further provides a rapid method of identifying a compound which inhibits apoptosis.

What is claimed is:  
     
         1 . A single-well, microscale method of determining the specific apoptotic activity of a cell comprising contacting a cell population of about 1×10 5  cells for a time period of between about 30 minutes and 4 hours with a sufficient volume of medium containing an apoptotic specific diagnostic reagent and a diagnostic accessory reagent so as to cover said cell population, and determining the activity of said apoptotic specific diagnostic reagent.  
     
     
         2 . The method of  claim 1 , wherein said cell population further comprises greater than about 10,000 cells, more preferably about 50,000 cells, and preferably about 100,000 cells.  
     
     
         3 . The method of  claim 1 , wherein said time period further comprises between about 30 minutes to 4 hours, preferably between about 30 minutes to 2 hours, and more preferably about 1 hour.  
     
     
         4 . The method of  claim 1 , wherein said volume further comprises between about 1 and 200 μl, preferably between about 30 and 125 μl, more preferably about 100 μl.  
     
     
         5 . The method of  claim 1 , wherein said apoptotic specific diagnostic reagent further comprises an caspase specific substrate or Annexin V.  
     
     
         6 . The method of  claim 5 , wherein said diagnostic accessory reagent is a lysis reagent or calcium.  
     
     
         7 . The method of  claim 1 , wherein said apoptotic specific diagnostic reagent further comprises an caspase specific substrate attached to a detectable label.  
     
     
         8 . The method of  claim 1 , wherein said apoptotic specific diagnostic reagent is selected from the group consisting of ZEVD-AMC, YVAD-AMC and DEVD-AMC.  
     
     
         9 . The method of  claim 1 , further comprising a multiwell format.  
     
     
         10 . The method of  claim 1 , further comprising a plurality of different single-wells for the simultaneous determination of multiple different samples.  
     
     
         11 . A method of identifying a compound which induces apoptosis comprising: 
 (a) providing a cell overexpressing a cell survival polypeptide, said cell survival polypeptide being overexpressed at a level which is sufficient to prevent the induction of apoptosis;    (b) treating said cell overexpressing said cell survival polypeptide with a direct stimulus of the cell death pathway;    (c) adding a compound to be tested for apoptotic inducing activity, and    (d) determining cellular apoptotic activity, wherein the presence of apoptotic activity is indicative of said compound being an apoptotic inducer.    
     
     
         12 . The method of  claim 11 , wherein said cell survival polypeptide comprises Bcl-2, Bcl-xL, Mcl-1 and E1B-19K.  
     
     
         13 . The method of  claim 11 , wherein said cell survival polypeptide is overexpressed at a level so as to prevent the induction of a direct stimulus of the cell death pathway.  
     
     
         14 . The method of  claim 13 , wherein said cell survival polypeptide is overexpressed at a level so as to prevent the induction of apoptosis by a direct stimulus selected from the group consisting of Fas ligand, anti-Fas antibody, staurosporine, UV and gamma irradiation.  
     
     
         15 . The method of  claim 11 , wherein said direct stimulus of the cell death pathway is selected from the group consisting of Fas ligand, anti-Fas antibody, staurosporine, UV and gamma irradiation.  
     
     
         16 . The method of  claim 11 , wherein said cell survival polypeptide is encoded by an exogenous nucleic acid.  
     
     
         17 . The method of  claim 16 , wherein said exogenous nucleic acid is either a homologous or heterologous nucleic acid.  
     
     
         18 . The method of  claim 11 , wherein said cell survival polypeptide is encoded by an endogenous nucleic acid.  
     
     
         19 . The method of  claim 11 , wherein said compound tested for apoptotic inducing activity further comprises a compound which induces caspase activity in a cell.  
     
     
         20 . The method of  claim 11 , wherein said compound tested for apoptotic inducing activity further comprises a compound which inhibits the activity of a cell survival polypeptide.  
     
     
         21 . The method of  claim 11 , wherein said compound tested for apoptotic inducing activity further comprises a compound which promotes the activity of a cell death polypeptide.  
     
     
         22 . The method of  claim 11 , wherein said apoptotic activity in step (d) further comprises lysing said cells and determining the caspase activity in said lysate.  
     
     
         23 . The method of  claim 11 , wherein said apoptotic activity in step (d) further comprises contacting the cells with Annexin 5 and determining the amount of Annexin 5 which binds.  
     
     
         24 . A rapid method of identifying a compound which inhibits apoptosis comprising: 
 (a) separately contacting a plurality of cell populations with a different compound to be tested for apoptotic inhibiting activity;    (b) incubating said cells with a direct stimulus of the cell death pathway for a period of between about 2 minutes to 3 hours, and    (c) measuring the specific apoptotic activity of the cells.    
     
     
         25 . The method of  claim 24 , wherein said direct stimulus of the cell death pathway is selected from the group consisting of Fas ligand, anti-Fas antibody and staurosporine UV and gamma irradiation.  
     
     
         26 . The method of  claim 24 , wherein step (c) further comprises lysing said cells and determining caspase activity in said lysate.  
     
     
         27 . The method of  claim 24 , wherein said compound exhibits caspase inhibitory activity.  
     
     
         28 . The method of  claim 24 , wherein said compound promotes the activity of a cell survival polypeptide.  
     
     
         29 . The method of  claim 24 , wherein said compound exhibits cell death polypeptide inhibitory activity.