SEPARATE METABOLIC PATHWAYS LEADING TO DNA FRAGMENTATION AND APOPTOTIC CHROMATIN CONDENSATION

Apoptosis is the predominant form of cell death observed in a variety of physiological and pathological conditions such as cancer involution , insect metamorphosis, the development of the immune and nervous syst ems, and embryogenesis. The typical nuclear changes taking place in ap optotic cells include extensive condensation of chromatin and internuc leosomal DNA fragmentation into units of 200 base pairs. However, the mechanisms responsible for both chromatin condensation and DNA fragmen tation have yet to be elucidated. In this study, micrococcal nuclease and the divalent cations, Ca2+ and Mg2+, were applied to isolated nucl ei in an attempt to reconstitute in vitro the digestion of genomic DNA associated with apoptosis. Micrococcal nuclease was found to induce a typical pattern of DNA fragmentation, but did not give rise to chroma tin condensation, whereas Ca2+/Mg2+ induced both chromatin condensatio n and DNA fragmentation in isolated mouse liver nuclei. When the endon uclease inhibitor ZnCl2 was used, the DNA fragmentation induced by Ca2 +/Mg2+ in nuclei could be completely inhibited, but chromatin condensa tion still occurred. For comparison, intact liver cells were treated w ith valinomycin, a potassium ionophore, which gave rise to an atypical cell death, with chromatin condensation appearing without DNA fragmen tation. Our results suggest that endonuclease activation in apoptosis is neither necessary nor sufficient to induce chromatin condensation, and that DNA fragmentation and chromatin condensation may be triggered through separate pathways during apoptosis.