3,4-DICHLOROISOCOUMARIN SERINE-PROTEASE INHIBITOR INDUCES DNA FRAGMENTATION AND APOPTOSIS IN SUSCEPTIBLE TARGET-CELLS

3,4-Dichloroisocoumarin (DCI) inhibition of serine proteases generates reactive intermediates that have been theorized to affect apoptosis, To examine this possibility various target cells were treated with dif ferent concentrations of DCI and assessed for intracellular nuclear DN A fragmentation and apoptosis, DCI treatment caused oligonucleosomal D NA fragmentation in cell lines expressing high levels of protease acti vity (LAK cells, NK-92, CTLL-2, L929, 3T3). This DNA breakdown charact eristic of apoptosis occurred in a dose-dependent fashion within 4-6 h r of treatment and was confirmed by electron microscopy. In cell lines expressing low levels of protease activity (unstimulated human periph eral blood mononuclear (PBMN) cells, YAC-1 cells), DCI effectively inh ibited protease activity without inducing oligonucleosomal DNA fragmen tation. ZN(2+) ions significantly inhibited DCI-induced DNA degradatio n. The mixture of DCI and BLT esterase active NK cell lysate triggered DNA fragmentation in isolated YAC-1 nuclei. Degree of DNA fragmentati on in YAC-1 nuclei was proportional to the level of BLT esterase activ ity, Cell lysate protease activity, initially inhibited by DCI acylati on, was restored by hydroxylamine deacylation, thus preventing DCI-med iated DNA fragmentation. Our results suggest that DCI treatment of cel ls expressing high levels of protease activity generates toxic levels of acyl-enzyme intermediates. These intermediates may trigger nuclear DNA breakdown and apoptosis by activating endogenous endonucleases, Th is effect may compromise the analysis of apoptosis in experimental sys tems using high concentrations of DCI for extended periods.