DNA strand breaks occurring during apoptosis - their early insitu detection by the terminal deoxynucleotidyl transferase and nick translation assays and prevention by serine protease inhibitors

Abstract

The appearance of DNA strand breaks during apoptosis was detected in individual cells, in relation to the cell cycle phase, by a novel assay based on labeling 3'-OH termini with biotinylated dUTP using exogenous terminal transferase or DNA polymerase. Apoptosis was induced in HL-60 cells by the DNA topoisomerase I and II inhibitors, and in rat thymocytes by prednisolone. Formation of strand breaks was prevented by the serine protease irreversible inhibitors diisopropyl fluorophosphate (DFP), L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK), N-p-tosyl-L-lysine chloromethyl ketone (TLCK) and by the substrates N-alpha-tosyl-L-arginine methyl ester (TAME) and N-benzoyl-L-tyrosine ethyl ester (BTEE). The data indicate that initiation of DNA degradation during apoptosis is preceded by a proteolytic step and suggest that apoptosis starts with activation (e.g. by DNA lesions) of a serine protease which hydrolyses protein(s) associated with the internucleosomal linker DNA sections, thus increasing accessibility of linker DNA to the apoptosis-associated endonuclease.