proteaseinhibitorsdid not preventthe cleavageof chromatinby exoge

Cleavage of cellular DNA into high molecular weight (predominantly 50 kb) fragments is an early event during apoptosis. We previously reported that this fragmentation was a Ca2'-independent process during apoptosis, which was induced by anticancer agents in human leukemia cells. The present study demonstrated that a high molecular weight DNA fragmentation activity (HDFA) was induced in the drug-treated cells and, uponfusionofthe drug-treatedcellswithuntreatedtargetcellsprelabeled with t'4Clthymidine, caused fragmentation of the labeled DNA in the target cells. Furthermore, extracts of the drug-treated cells caused high molecular weight DNA fragmentation In nuclei isolated from untreated cells. Biochemical characterization of HDFA revealed the following prop erties: HDFA was proteinaceous in nature, as evidenced by its inactivation by heating or by digestion with protelnase K; HDFA required Mg@@for optimal activity but was inhibited by Zn2@ and K@; HDFA was active in vitro at pH 6.0—8.0and was inactive under more acidic conditions (pH < 6.0); addition of ATP (0.5-2 mM)substantially potentiated HDFA activity in isolated nuclei; and HDFA was not inhibited by actin (an inhibitor of DNase I) but was inhibited by the extracts from K562 cells, which were resistant to drug-induced apoptosis. The specific inhibitor of cystelne proteases (Interleukin lfi-converting enzyme protease family) blocked the generation of drug-induced high molecular weight DNA fragmentation in whole cells, whereas in isolated nuclei, the cysteine proteaseinhibitorsdid not preventthe cleavageof chromatinby exoge nous HDFA. These resWts suggest that, once HDFA is activated during apoptosis, it does not require the presence of cystelne proteases for its endonucleolytic activity and that the cysteine proteases may be involved in the apoptoticprocessupstreamof the activationof HDFAin wholecells.