Extent and Limitation of the Control of Nuclear Apoptosis by DNA-Fragmenting Factor

Abstract

During apoptosis, changes to the nucleus of the dying cell include DNA degradation and structural collapse. These changes are accomplished by caspase-mediated cleavage of DNA-fragmenting factor DFF₄₅, an inhibitor of the effector molecule DFF₄₀. DFF₄₅and, more efficiently, a mutant lacking one caspase-cleavage site (DFF₄₅m) inhibited nuclear changes in a cell-free system when apoptosis was initiated by adding caspase-3 to cell extracts. In primary tissues from several mammalian species, human caspase-3 activated and human DFF₄₅m blocked nuclear apoptosis demonstrating evolutionary conservation of this step. However, DFF₄₅m did not significantly inhibit DNA-fragmenting activity in extracts from staurosporine-treated cells from the human cell line Jurkat. In extracts from normal Jurkat cells, DFF₄₅m blocked caspase-triggered DNA cleavage efficiently only if added within a short time of the addition of the caspase. At later time points, this inhibition by DFF₄₅m was strongly reduced in efficiency while Zn²⁺still completely blocked DNA fragmentation. These results demonstrate the evolutionary conservation of a linear pathway in apoptosis and suggest the existence of more complex events as final effector machinery.