Double and triple readout assay systems

Abstract

The present invention provides assays for identifying compounds that affect the transcriptional activity of a protein of interest or affect the stability of the protein of interest. The triple readout assay system which can be used to identify compounds that affect the transcriptional activity of a protein of interest uses three cell lines to control for non-specific effects such as sequences flanking the inserted gene and cytotoxicity. The double readout assay system assesses protein stability and uses a fusion protein of a reporter and the protein of interest. These assay systems may be particularly useful in identifying compounds that affect transcription factors and tumor suppressors. In a particular embodiment, the tumor suppressor p53 is the target protein being studied.

What is claimed is:  
     
         1 . A method of identifying compounds that affect p53 stability, the method comprising the steps of: 
 providing a cell line transfected with a construct expressing p53-fused to a reporter protein and a control reporter protein;    contacting a test compound with cells from the cell line;    contacting cells with a DNA damaging agent; and    comparing levels of the p53 fusion protein and the control reporter protein.    
     
     
         2 . A method of identifying compounds that affect p53 stability, the method comprising the steps of: 
 providing a cell line transfected with a construct expressing p53-fused to a reporter protein and a control reporter protein, and transfected to express E6;    contacting a test compound with cells from the cell line;    contacting cells with a DNA damaging agent; and    comparing levels of the p53 fusion protein and the control reporter protein.    
     
     
         3 . The method of  claim 1  wherein the cell line is derived from a cell line known to contain active p53 turnover pathways.  
     
     
         4 . The method of  claim 1  wherein the cell line is derived from a cell line that has a relatively low steady state level of p53 protein and shows a significant accumulation of p53 protein following treatment with a DNA damaging agent.  
     
     
         5 . The method of  claim 1  wherein the DNA damaging agent is a chemotherapeutic agent.  
     
     
         6 . The method of  claim 1  wherein the DNA damaging agent is adriamycin.  
     
     
         7 . The method of  claim 1  wherein the cell line is an animal cell line.  
     
     
         8 . The method of  claim 1  wherein the cell line is a mammalian cell line.  
     
     
         9 . The method of  claim 1  wherein the cell line is a human cell line.  
     
     
         10 . The method of  claim 1  wherein the cell line is an RKO cell line.  
     
     
         11 . The method of  claim 1  wherein the cell line is an MCF7 cell line.  
     
     
         12 . The method of  claim 1  wherein the p53 fusion protein is detectable by an antibody.  
     
     
         13 . The method of  claim 1  wherein the control reporter protein is detectable by an antibody.  
     
     
         14 . The method of  claim 1  wherein the p53 fusion protein is detectable by a change in absorbance, change in fluorescence, or radio-immuno assay.  
     
     
         15 . The method of  claim 1  wherein the control reporter protein is detectable by a change in absorbance, change in fluorescence, or a radio-immuno assay  
     
     
         16 . The method of  claim 1  wherein the p53 fusion protein and the control reporter protein are translated from a single mRNA transcript.  
     
     
         17 . The method of  claim 16  wherein an internal ribosome entry site is inserted between a gene encoding the p53 fusion protein and a gene encoding the control reporter protein.  
     
     
         18 . The method of  claim 1  wherein a gene encoding the p53 fusion protein and a gene encoding the control reporter protein are controlled by a same promoter sequence and regulatory sequences.  
     
     
         19 . The method of  claim 1  wherein the reporter protein or the control reporter protein is selected from the group consisting of firefly luciferase, secreted alkaline phosphatase, enhanced green fluorescent protein, horseradish peroxidase, beta-galactosidase, and renilla luciferase.  
     
     
         20 . The method of  claim 1  wherein the reporter protein or the control reporter protein is selected from the group consisting of firefly luciferase and renilla luciferase.  
     
     
         21 . The method of  claim 1  wherein the p53 fusion protein and the control reporter protein are transcribed at substantially the same level.  
     
     
         22 . The method of  claim 1  wherein the construct comprises a gene encoding a p53/FL (firefly luciferase) fusion protein, and a gene encoding RL (renilla luciferase) protein.  
     
     
         23 . The method of  claim 22  wherein the construct further comprises a CMV promoter controlling the expression of the gene encoding the p53/FL fusion protein and the gene encoding the RL protein, and an internal ribosome entry site.  
     
     
         24 . A kit useful in identifying compounds that affect p53 stability comprising: 
 a cell line with a construct expressing p52-fused to a reporter protein and a control reporter protein.    
     
     
         25 . A method of identifying compounds that affect stability of a protein of interest, the method comprising steps of: 
 providing a cell line transfected with a construct expressing a first reporter protein fused to the protein of interest, and a second control reporter protein;    contacting a test compound with cells from the cell line;    contacting the cells with a DNA damaging agent; and    comparing levels of the first reporter protein and the second reporter protein.    
     
     
         26 . A method of identifying compounds that affect stability of a protein of interest, the method comprising steps of: 
 providing a cell line transfected with a construct expressing a first reporter protein fused to the protein of interest, and a second control reporter protein;    contacting a test compound with cells from the cell line;    contacting cells with a DNA damaging agent; and    comparing levels of the first reporter protein and the second reporter protein.