3,4-Dichloroisocoumarin Serine Protease Inhibitor Induces DNA Fragmentation and Apoptosis in Susceptible Target Cells

Abstract

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 different concentrations of DCI and assessed for intracellular nuclear DNA fragmentation and apoptosis. DCI treatment caused oligonucleosomal DNA fragmentation in cell lines expressing high levels of protease activity (LAK cells, NK-92, CTLL-2, L929, 3T3). This DNA breakdown characteristic of apoptosis occurred in a dose-dependent fashion within 4-6 hr of treatment and was confirmed by electron microscopy. In cell lines expressing low levels of protease activity (unstimulated human peripheral blood mononuclear (PBMN) cells, YAC-1 cells), DCI effectively inhibited protease activity without inducing oligonucleosomal DNA fragmentation. ZN⁺ ions significantly inhibited DCl-induced DNA degradation. The mixture of DCI and BLT esterase active NK cell lysate triggered DNA fragmentation in isolated YAC-1 nuclei. Degree of DNA fragmentation in YAC-1 nuclei was proportional to the level of BLT esterase activity. Cell lysate protease activity, initially inhibited by DCI acylation, was restored by hydroxylamine deacylation, thus preventing DCi-mediated DNA fragmentation. Our results suggest that DCI treatment of cells expressing high levels of protease activity generates toxic levels of acylenzyme intermediates. These intermediates may trigger nuclear DNA breakdown and apoptosis by activating endogenous endonucleases. This effect may compromise the analysis of apoptosis in experimental systems using high concentrations of DCI for extended periods.