SEPARATE POOLS OF ENDONUCLEASE ACTIVITY ARE RESPONSIBLE FOR INTERNUCLEOSOMAL AND HIGH-MOLECULAR-MASS DNA FRAGMENTATION DURING APOPTOSIS

The oligonucleosomal pattern of DNA fragmentation is the best-characte rized biochemical marker of apoptosis and believed to be generated by a, as yet unidentified, Ca2+, Mg2+-dependent endonuclease. All apoptot ic cells fragment their genome. However, not every cell type undergoin g apoptosis is capable of internucleosomal DNA cleavage. We have analy zed the endonuclease activities and patterns of DNA fragmentation in f our established cell lines undergoing apoptosis following serum depriv ation, i.e., rat 5123tc hepatoma and PC12 pheochromocytoma, as well as human MCF7 breast and DU145 prostatic carcinoma cells. Whereas apopto tic 5123tc and PC12 cells degraded their DNA into oligonucleosomes, th e MCF7 and DU145 cells generated only >50 kilobase pairs (kbp) DNA fra gments. However, when isolated nuclei from all four cell lines were in cubated with both Ca2+ and Mg2+ ions, their DNA was cleaved into inter nucleosomal fragments. Following washing with a low ionic strength buf fer, the nuclei could only degrade DNA to >50-kbp fragments. DNA ladde rs were produced again in these washed nuclei after reconstitution wit h the nuclear wash, which contained an endonucleolytic activity of app roximately 97 kilodaltons. These experiments showed that cells maintai n separate pools of endonucleolytic activities responsible for the hig h and low molecular mass DNA fragmentation, and depending on the cell type, one or both enzymatic pools become activated during apoptosis.