Methods for Detecting DNA Damage and Screening for Cancer Therapeutics

Abstract

A method for detecting DNA damage in a tissue sample involves contacting an immobilized biological sample with a labeled ligand which binds to human 53Bp1, and examining the immobilized sample for the presence of a label generated-detectable signal concentrated in foci in said sample. The presence of concentrated foci is indicative of DNA damage and the presence of diffuse signal is indicative of a normal sample. Diagnostic reagents contain a ligand that binds to human 53Bp1 associated with a detectable label. Diagnostic kits for detecting DNA damage in a biological sample contain such diagnostic reagents and signal detection components. Compositions that inhibit or antagonize the biological activity of 53Bp1 are identified by suitable assays, and are employed in methods of retarding the growth of a cancer cell.

1 . A method for detecting DNA damage in a tissue sample, comprising: 
 contacting a biological sample with a ligand which binds to human 53Bp1, said ligand associated with a label which provides a detectable signal;    examining said biological sample for the presence of signal concentrated in foci of 53Bp1 in said biological sample, wherein the presence of concentrated foci is indicative of DNA damage and the presence of diffuse signal is indicative of a normal sample.    
     
     
         2 . The method according to  claim 1 , further comprising immobilizing said biological sample prior to said contacting or examining.  
     
     
         3 . The method according to  claim 1 , wherein said biological sample is a white blood cell or a biopsy specimen.  
     
     
         4 . The method according to  claim 1 , wherein said ligand is selected from the group consisting of a polyclonal antibody, a monoclonal antibody or a recombinant antibody of classes IgG, IgM, IgA, IgD and IgE; a Fab, Fab=or F(ab=)2, or Fc antibody fragment thereof, a single chain Fv antibody fragment, a recombinant construct comprising a complementarity determining region of an antibody directed to 53Bp1, a synthetic antibody or chimeric antibody or humanized antibody construct which shares sufficient CDRs of an antibody to 53Bp1 to retain functionally equivalent binding characteristics of an antibody that binds 53Bp1.  
     
     
         5 . The method according to  claim 1 , wherein said examining comprises performing immunofluorescent microscopy or immunohistochemical analysis with a suitable ligand.  
     
     
         6 . The method according to  claim 1 , wherein said 53Bp1 comprises a fragment comprising amino acid residues 1220 to 1711 of SEQ ID NO: 2.  
     
     
         7 . A diagnostic reagent comprising a ligand that binds to human 53Bp1, said ligand associated with a detectable label.  
     
     
         8 . The diagnostic reagent according to  claim 7 , which is an anti 53Bp1 monoclonal antibody.  
     
     
         9 . The diagnostic reagent according to  claim 7 , wherein said 53Bp1 comprises a fragment comprising amino acid residues 1220 to 1711 of SEQ ID NO: 2.  
     
     
         10 . A diagnostic kit for detecting DNA damage in a biological sample, comprising said diagnostic reagent of  claim 7 .  
     
     
         11 . A composition that antagonizes or inhibits the biological activity or expression of 53Bp1, or binds to 53Bp1.  
     
     
         12 . A method of screening test compounds to identify a composition of  claim 11 , comprising employing a 53Bp1 ligand associated with a detectable label to detect the expression of 53Bp1 in a cell contacted with a test compound or to detect the presence or number of 53Bp1 induced nuclear foci in cells contacted with a test compound.  
     
     
         13 . The method according to  claim 12 , further comprising: 
 (a) providing a first cell which is contacted with a suitable amount of a test compound;    (b) providing a control comprising a cell identical to said first cell but without exposure to said test compound;    (c) exposing said cells (a) and (b) to a DNA damaging agent;    (d) contacting said cells (c) with a ligand that binds 53Bp1, said ligand associated with a detectable label;    (e) examining said cells (d) for detection of the signal generated by said label, which signal indicates the presence and number of 53Bp1 nuclear foci, and    (f) comparing the signals generated by the labels in said two cells,    wherein a lesser detectable signal in said cell (a) compared with the detectable signal in said cell (b) indicates that said test compound has inhibited the presence and/or number of 53Bp1 foci in cell (a) and is a 53Bp1 inhibitor.    
     
     
         14 . A method of retarding the growth of a cancer cell, comprising administering a composition of  claim 11  to the site of a cancer cell.  
     
     
         15 . The method according to  claim 14 , comprising administering said composition to a cell ex vivo or to a mammal having a cancer in vivo.  
     
     
         16 . A method of targeting a tumor cell for delivery of a therapeutic agent, comprising administering to a patient bearing a tumor containing 53Bp1 foci a ligand that binds to 53Bp1, said ligand associated with a compound that retards the growth of, or kills, said tumor cell.  
     
     
         17 . The method according to  claim 16 , wherein said compound is selected from the group consisting of a radionucleotide, a toxin, a bi-specific antibody and an anticancer drug optionally linked to a protein or peptide.  
     
     
         18 . The method according to  claim 16 , wherein said ligand binds a fragment of 53Bp1 comprising amino acid residues 1220 to 1711 of SEQ ID NO: 2.