ROLE OF PROTEOLYSIS IN APOPTOSIS - INVOLVEMENT OF SERINE PROTEASES ININTERNUCLEOSOMAL DNA FRAGMENTATION IN IMMATURE THYMOCYTES

Three chemically distinct serine, but not cysteine, protease inhibitor s (phenylmethylsulphonyl fluoride, N-tosyl-L-phenylalanylchloromethyl ketone and 3,4-dichloroisocoumarin) prevented, in a dose-dependent man ner, the characteristic apoptotic internucleosomal DNA cleavage (DNA l adder) typically observed in thymocytes in response to dexamethasone a nd teniposide VM-26. This effect was not the result of a direct inhibi tion of the Ca2+, Mg2+ -dependent endonuclease, since oligonucleosomal DNA cleavage occurred in the presence of these inhibitors in isolated nuclei. The proteolytic step occurred at a very early stage of apopto sis, and preincubation of thymocytes with the inhibitors before dexame thasone or teniposide VM-26 were added irreversibly suppressed ladder formation. This implied that the cellular effector(s) of these compoun ds preexisted and were not resynthesized in response to the inducers o f apoptosis. Serine protease inhibitors also suppressed apoptotic cell shrinkage and complete nuclear collapse, suggesting that these morpho logical changes were directly related to internucleosomal fragmentatio n of DNA. However, the serine protease inhibitors did not prevent high molecular weight DNA cleavage (>50 kilobases) that preceded the ladde r formation and thymocytes still died by apoptosis. This supported the view that internucleosomal DNA cleavage, considered to be the biochem ical marker of apoptosis, might in fact be a late and dispensable step and that the newly described high molecular weight DNA cleavage might be a better indicator of apoptosis.