Method for determining activity of cell cycle regulatory factor and method for diagnosing cancer using the same

Abstract

A method for determining the activity of a cell cycle regulatory factor comprising the steps of preparing a sample for measuring a cyclin-dependent kinase/cyclin complex from living cells; reacting adenosine 5′-O-( 3 -thiotriphosphate) (ATP-γ S) with a substrate for the cyclin-dependent kinase in presence of the sample in order to introduce a monothiophosphate group into a serine or threonine residue of the substrate; labeling the substrate by coupling a labeling fluorophore or a labeling enzyme with a sulfur atom of the introduced monothiophosphate group; measuring the amount of fluorescence from the labeling fluorophore labeling the substrate, or reacting the labeling enzyme labeling the substrate with a substance which generates an optically detectable product by reaction with the labeling enzyme and optically measuring the amount of the generated product; and calculating the activity of the cyclin-dependent kinase from the measured amount of fluorescence or the measured amount of the generated product with reference to a pre-produced reference curve.

1 . A set of reagents for measuring the activity of cyclin-dependent kinase in a sample prepared from a living cell, which comprises: 
 an anti-cyclin-dependent kinase antibody,    a substrate for cyclin-dependent kinase,    adenosine 5′-O-(3-thiotriphosphate) (ATP-γS); and    a labeling fluorophore or a labeling enzyme for coupling with a reaction product of the substrate and the ATP-γS.    
   
   
       2 . The set of reagents according to  claim 1 , wherein the cyclin-dependent kinase is selected from the group consisting of CDK1, CDK2, CDK4 and CDK6.  
   
   
       3 . The set of reagents according to  claim 1 , further comprising a lysis buffer for solubilizing the living cell.  
   
   
       4 . The set of reagents according to  claim 3 , wherein the lysis buffer contains a surfactant, a protease inhibitor and a phosphatase inhibitor.  
   
   
       5 . The set of reagents according to  claim 1 , wherein the cyclin-dependent kinase is CDK1 or CDK2 and the substrate is histone H1.  
   
   
       6 . The set of reagents according to  claim 1 , wherein the cyclin-dependent kinase is CDK4 or CDK6 and the substrate is Rb whose cysteine residue is substituted by an amino acid residue which does not contain thiol residue.  
   
   
       7 . The set of reagents according to  claim 1 , wherein the labeling fluorophore is a fluorescent dye.  
   
   
       8 . The set of reagents according to  claim 7 , wherein the fluorescent dye is FITC.  
   
   
       9 . The set of reagents according to  claim 1 , wherein the labeling enzyme is peroxidase.  
   
   
       10 . The set of reagents according to  claim 1 , further comprising a coupling reaction stopping agent for stopping a coupling reaction between the labeling fluorophore or the labeling enzyme and the reaction product.  
   
   
       11 . The set of reagents according to  claim 10 , wherein the coupling reaction stopping agent comprises a thiol.  
   
   
       12 . A combination of reagents for measuring activity of cyclin-dependent kinase in a sample prepared from a living cell, which comprises: 
 an anti-cyclin-dependent kinase antibody,    a substrate for cyclin-dependent kinase,    adenosine 5′-O-(3-thiotriphosphate) (ATP-γS); and    a labeling fluorophone or a labeling enzyme for coupling with a reaction product of the substrate and the ATP-γS.    
   
   
       13 . The combination of reagents according to  claim 12 , further comprising a lysis buffer for solubilizing the living cell.  
   
   
       14 . The combination of reagents according to  claim 13 , wherein the lysis buffer contains a surfactant, a protease inhibitor and a phosphatase inhibitor.  
   
   
       15 . The combination of reagents according to  claim 12 , wherein the further comprising a coupling reaction stopping agent for stopping a coupling reaction between the labeling fluorophore or the labeling enzyme and the reaction product.  
   
   
       16 . The combination of reagents according to  claim 15 , wherein the coupling reaction stopping agent comprises a thiol.