ENDO-EXONUCLEASE OF HUMAN LEUKEMIC-CELLS - EVIDENCE FOR A ROLE IN APOPTOSIS

Inactive forms of endo-exonuclease, activated in vitro by treatment wi th trypsin, have been identified in human leukaemic CEM, and MOLT-4 ce lls. They comprise over 95% of the total single-strand DNase activity in nuclei and are mainly bound to chromatin and the nuclear matrix, Th e activated enzyme had Mg2+(Mn2+)-dependent, Ca2+-stimulated activitie s with single- and double-strand DNAs and RNA (polyriboadenylic acid) and other properties characteristic of endo-exonucleases previously de scribed, At least twice as much inactive endo-exonuclease has also bee n localised in extranuclear compartments of CEM and MOLT-4 cells, 85% bound to the membranes of the endoplasmic reticulum and 15% free in th e cytosol. The soluble cytosolic trypsin-activatable endo-exonuclease was immunoprecipitated by antibodies raised independently to both Neur ospora and monkey CV-1 cell endo-exonucleases, The free and bound enzy mes of both nuclear and extranuclear compartments also cross-reacted o n immunoblots with the antibody raised to Neurospora endo-exonuclease to reveal multiple polypeptides ranging in size from 18 to 145 kDa, ma ny of which exhibited activity on DNA gels, The major species bound to the chromatin/matrix were in the 55-63 kDa range, Limited proteolysis of the large polypeptides to those of 18 to 46 kDa accompanied sponta neous chromatin DNA fragmentation to form DNA 'ladders' in an isolated nuclei/cytosol system. When the leukaemic cells were treated in cultu re with either etoposide or podophyllotoxin to induce apoptosis, the l argest polypeptides disappeared and smaller endo-exonuclease-related p olypeptides of 18 to 46 kDa were detected in the nuclear extracts, The appearance of these polypeptides also correlated with extensive chrom atin DNA fragmentation, In addition, there were correlations between t he depletion of the major 55-63 kDa species bound to the membranes of the endoplasmic reticulum, depletion of the extranuclear trypsin-activ atable activity and the onset and extent of chromatin DNA fragmentatio n in both cell lines, The extranuclear 55-63 kDa species may be precur sors of the chromatin/matrix bound endo-exonuclease. The results indic ate that endo-exonuclease plays a role in chromatin DNA degradation in mammalian cells during apoptosis.