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 activity emerging during early apoptosis is inhibited during normal cell life. In a cell-free system, cytosol fractions from diverse nonapoptotic human cell lines (Jurkat T-cell leukemia, HeLa carcinoma, SK-N-MC neuroblastoma, and WI-38 embryonic lung fibroblast) potently neutralized the nuclear DNA fragmentation 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 healthy cytosol in inhibiting DNA fragmentation. An antiserum against human DFF45 detected 44 and 34 kDa proteins (major and minor, respectively) in the cytosols but not in the nuclear or membrane fractions of various cultured human 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 healthy cells.
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Laboratory of Biochemical Genetics, National Heart, Lung, and Blood Institute, Bethesda, Maryland 20892, USA. sabol@codon.nih.gov
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Abdul-Khalek
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D000230:Adenocarcinoma; D017209:Apoptosis / Q000187:drug effects; D051017:Apoptosis Regulatory Proteins; D002460:Cell Line; D002850:Chromatography, Gel; D053938:DNA Fragmentation / Q000187:drug effects; D003851:Deoxyribonucleases / Q000037:antagonists & inhibitors*; D004791:Enzyme Inhibitors / Q000276:immunology / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D007163:Immunosorbent Techniques; D019169:Jurkat Cells; D011506:Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000502:physiology*; D011994:Recombinant Proteins / Q000494:pharmacology; D013347:Subcellular Fractions / Q000737:chemistry; D014407:Tumor Cells, Cultured
D051017:Apoptosis Regulatory Proteins; D004791:Enzyme Inhibitors; D011506:Proteins; D011994:Recombinant Proteins; C109664:caspase-activated DNase inhibitor; D003851:Deoxyribonucleases