We are investigating the molecular mechanisms of apoptosis (programmed cell death), an important process of orderly cell suicide executed under a variety of physiological and pathological circumstances by a genetically controlled program of biochemical reactions. We have developed a cell-free system to study the nuclear events of apoptosis -- internucleosomal DNA fragmentation, chromatin condensation, and nuclear fragmentation. We have also developed a rapid filtration assay to quantitate apoptosis-associated DNA fragmentation occurring in the cell- free reactions. We have applied these techniques to Jurkat human T-cell leukemia cells undergoing apoptosis triggered by Fas antigen ligation by Fas antibodies. Within two hours of anti-Fas exposure, a cytosolic activity is generated that elicits, in the cell-free system, DNA fragmentation and morphological changes of apoptosis in nuclei purified from healthy Jurkat cells. This activity is associated with a heat- sensitive protein which we term """"""""apoptin."""""""" It is inhibited by several serine protease inhibitors and thiol reagents but not by inhibitors of ICE-family proteases or the proteosome, which are critically involved in apoptosis. We suggest that apoptin is a novel and important enzyme, possibly a protease, which is activated by upstream ICE-family protease(s) during apoptosis and which in turn participates in apoptotic execution by activating nuclear nuclease(s). We have also identified a protein inhibitor of apoptin in the cytosol of healthy Jurkat cells and other transformed cells. Our goals are to purify these proteins and further characterize them and their functions.
