Two distinct pathways leading to nuclear apoptosis

Apaf-1(-/-) or caspase-3(-/-) cells treated with a variety of apoptosis ind ucers manifest apoptosis-associated alterations including the translocation of apoptosis-inducing factor (AIF) from mitochondria to nuclei, large scal e DNA fragmentation, and initial chromatin condensation (stage I). However, when compared with normal control cells, Apaf-1(-/-) or caspase-3(-/-) cel ls fail to exhibit oligonucleosomal chromatin digestion and a more advanced pattern of chromatin condensation (stage II). Microinjection of such cells with recombinant AIF only causes peripheral chromatin condensation (stage I), whereas microinjection with activated caspase-3 or its downstream targe t caspase-activated DNAse (CAD) causes a more pronounced type of chromatin condensation (stage II). Similarly, when added to purified HeLa nuclei, AIF causes stage I chromatin condensation and large-scale DNA fragmentation, w hereas CAD induces stage II chromatin condensation and oligonucleosomal DNA degradation. Furthermore, in a cell-free system, concomitant neutralizatio n of AIF and CAD is required to suppress the nuclear DNA loss caused by cyt oplasmic extracts from apoptotic wild-type cells. In contrast, AIF depletio n alone suffices to suppress the nuclear DNA loss contained in extracts fro m apoptotic Apaf-1(-/-) or caspase-3(-/-) cells. As a result, at least two redundant parallel pathways may lead to chromatin processing during apoptos is. One of these pathways involves Apaf-1 and caspases, as well as CAD, and leads to oligonucleosomal DNA fragmentation and advanced chromatin condens ation. The other pathway, which is caspase-independent, involves AIF and le ads to large-scale DNA fragmentation and peripheral chromatin condensation.