Inhibition of apoptosis-associated DNA fragmentation activity in nonapoptotic cells: The role of DNA fragmentation factor-45 (DFF45/ICAD)

We have investigated the mechanism whereby nuclear DNA fragmentation activi ty emerging during early apoptosis is inhibited during normal cell life. In a cell free system, cytosol fractions from diverse non-apoptotic human cel l lines (Jurkat T-cell leukemia, HeLa carcinoma, SK-N-MC neuroblastoma, and WI-38 embryonic lung fibroblast) potently neutralized the nuclear DNA frag mentation activity of cytosol from apoptotic anti-Fas treated Jurkat cells. Recombinant human DNA fragmentation factor 45 kDa subunit (DFF45/ICAD), an inhibitor of the caspase-activated DNase DFF40/CAD, substituted for health y cytosol in inhibiting DNA fragmentation, An antiserum against human DFF45 detected 44 and 34 kDa proteins (major and minor, respectively) in the cyt osols but not in the nuclear or membrane fractions of various cultured huma n cells. Cytosols depleted of DFF45/ICAD by immunoadsorption had little or no inhibitor of nuclear DNA fragmentation activity and no caspase-activated DNA fragmentation activity. We conclude that immunoreactive DFF45/ICAD is the principal inhibitor of apoptotic DNase activity in the cytosol of healt hy cells, (C) 1998 Academic Press.