Regulation of apoptosis and in vitro model for studies thereof

Abstract

A cell-free system based on the cytosol of normally growing cells, which reproduces measurable aspects of the apoptotic program, is provided. The apoptotic program is initiated by addition of dATP in the specific exemplification of the HeLa 100,000×g supernatant. Fractionation of the cytosol yielded a 15 kDa protein, identified by absorption spectrum and protein sequence as cytochrome c, that is required for in vitro apoptosis. Elimination of cytochrome c from cytosol by immunodepletion or inclusion of sucrose to stabilize mitochondria during cytosol preparation, diminished the apoptotic activity. Addition of exogenous cytochrome c to cytochrome c-depleted extracts restored apoptotic activity. Cells undergoing apoptosis in vivo showed increased release of cytochrome c to their cytosol, suggesting that mitochondria may function in apoptosis by releasing cytochrome c.

We claim:  
     
         1 . A method for assaying compositions in vitro for regulation of initiation of apoptosis, said method comprising the steps of: 
 (a) preparing a 100,000×g supernatant extract from cells derived from a multicellular eukaryote, which cells are not physiologically committed to apoptosis;    (b) introducing into an aliquot of the 100,000×g supernatant extract of step (a) a composition which can have a negative, positive or no effect on apoptosis to produce an extract assay,    (c) preparing control extract assays comprising, separately, a composition known to inhibit apoptosis, a composition known to induce apoptosis and a composition known to have no effect on apoptosis;    (d) in the alternative, assessing the activation of apoptosis in response to the introduction of the composition by determining an increase in cytosolic cytochrome c, an increase in CPP32 protease activity or increase in ability to fragment genomic DNA in nuclei introduced into the assay mixture as compared to increase in soluble cytochrome c, CPP32 protease activity or ability to fragment genomic DNA as compared to an assay lacking said composition; or assessing inhibition of activation of apoptosis in response to introduction of the composition into an assay mixture in the presence of a composition known to induce the apoptotic pathway in said 100,000×g extract by the reduction in soluble cytochrome c, CPP32 protease activity or genomic DNA fragmentation in an assay comprising said composition and a known inducer of the apoptotic pathway as compared to an assay comprising a known inducer of the apoptotic pathway but lacking said composition; and 
 comparing soluble cytochrome c, CPP32 protease activity or DNA fragmentation in an extract comprising said composition with soluble cytochrome c, CPP32 protease activity or DNA fragmentation in an extract lacking said composition;  
 whereby an apoptosis-inhibiting composition is identified by its ability to increase soluble cytochrome c, CPP32 protease activity or DNA fragmentation in the mammalian 100,000×g extract of step (a) and whereby an apoptosis-inhibiting composition is identified by its ability to inhibit induction of apoptosis in response to a known inducer of apoptosis in the mammalian 100,000×g extract of step (a) or whereby a composition with no effect on the induction of apoptosis is identified as having no effect on soluble cytochrome c, CPP32 protease activity or DNA fragmentation in the presence or absence of a known inducer of apoptosis.  
   
     
     
         2 . The method of  claim 1  wherein said cells are mammalian cells.  
     
     
         3 . The method of  claim 2  wherein cytosolic cytochrome c is detected in an immunological assay for cytochrome c.  
     
     
         4 . The method of  claim 2  wherein CPP32 protease activity is detected by introducing radiolabeled poly adenosine diphosphate-ribose polymerase (PARP) or one or more radiolabeled sterol regulatory binding proteins (SREBP) into the assay and subsequently detecting fragments of said PARP or SREBP in the assay.  
     
     
         5 . The method of  claim 4  wherein said SREBP is SREBP-2.  
     
     
         6 . The method of  claim 4  wherein fragments of PARP or SREBP are detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.  
     
     
         7 . The method of  claim 2  wherein DNA fragmentation is detected by introducing intact mammalian cell nuclei into the extract assays of steps (b) and (c), incubating and subsequently extracting genomic DNA and analyzing size distributions of said genomic DNAs to assess DNA fragmentation.  
     
     
         8 . The method of  claim 2  wherein the mammalian cells from which the 100,000×g extract is made are tumor cells and wherein compositions are tested for their ability to induce apoptosis.  
     
     
         9 . The method of  claim 8  wherein the tumor cells express a Bcl-2 protein and wherein compositions comprise chemotherapeutic agents tested for the ability to induce apoptosis despite presence of the Bcl-2 protein.  
     
     
         10 . The method of  claim 2  wherein the extract assays of steps (b) and (c) comprise deoxyadenosine triphosphate and/or deoxyadenosine diphosphate in an amount sufficient to allow the induction of the apoptotic response when assessing the inhibition of activation of apoptosis by the composition.  
     
     
         11 . The method of  claim 2  wherein said cells are HeLa cells.  
     
     
         12 . The method of  claim 10  wherein cytosolic cytochrome c is detected in an immunological assay for cytochrome c.  
     
     
         13 . The method of  claim 10  wherein CPP32 protease activity is detected by introducing radiolabeled poly adenosine diphosphate-ribose polymerase (PARP) or one or more radiolabeled sterol regulatory binding proteins (SREBP) into the assay and subsequently detecting fragments of said PARP or SREBP in the assay.  
     
     
         14 . The method of  claim 13  wherein said SREBP is SREBP-2.  
     
     
         15 . The method of  claim 13  wherein fragments of PARP or SREBP are detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.  
     
     
         16 . The method of  claim 10  wherein DNA fragmentation is detected by introducing intact mammalian cell nuclei into the extract assays of steps (b) and (c), incubating and subsequently extracting genomic DNA and analyzing size distributions of said genomic DNAs to assess DNA fragmentation.