DNA fragmentation induced by protease activation in p53-null human leukemia HL60 cells undergoing apoptosis following treatment with the topoisomerase I inhibitor camptothecin: cell-free system studies.
We studied the role of proteases in apoptosis using a cell-free system prepared from a human leukemia cell line. HL60 cells are p53 null and extremely sensitive to a variety of apoptotic stimuli including DNA damage induced by the topoisomerase I inhibitor, camptothecin. We measured DNA fragmentation induced in isolated nuclei by cytosolic extracts using a filter elution assay. Cytosol from camptothecin-treated HL60 cells induced internucleosomal DNA fragmentation in nuclei from untreated cells. This fragmentation was suppressed by serine protease inhibitors. Serine proteases (trypsin, endoproteinase Glu-C, chymotrypsin A, and proteinase K) and papain by themselves induced DNA fragmentation in naive nuclei. This effect was enhanced in the presence of cytosol from untreated cells. Cysteine protease inhibitors (E-64, leupeptin, Ac-YVAD-CHO [ICE inhibitor]) did not affect camptothecin-induced DNA fragmentation. The apopain/Yama inhibitor, Ac-DEVD-CHO, and the proteasome inhibitor, MG-132, were also inactive both in the cell-free system and in whole cells. Interleukin-1 beta converting enzyme (ICE) or human immunodeficiency virus protease failed to induce DNA fragmentation in naive nuclei. Together, these results suggest that DNA damage activates serine protease(s) which in turn activate(s) nuclear endonuclease(s) during apoptosis in HL60 cells.
Shimizu
T
T
Laboratory of Molecular Pharmacology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Pommier
Y
Y
D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D002166:Camptothecin / Q000494:pharmacology*; D002457:Cell Extracts; D002467:Cell Nucleus; D002474:Cell-Free System; D002843:Chromatin / Q000187:drug effects; D003546:Cysteine Endopeptidases / Q000378:metabolism / Q000494:pharmacology; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003600:Cytosol; D004247:DNA / Q000378:metabolism*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016333:HIV Protease / Q000494:pharmacology; D018922:HL-60 Cells; D006358:Hot Temperature; D006801:Humans; D012697:Serine Endopeptidases / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013997:Time Factors; D059004:Topoisomerase I Inhibitors; D016159:Tumor Suppressor Protein p53 / Q000502:physiology
D002457:Cell Extracts; D002843:Chromatin; D015853:Cysteine Proteinase Inhibitors; D004791:Enzyme Inhibitors; D015842:Serine Proteinase Inhibitors; D059004:Topoisomerase I Inhibitors; D016159:Tumor Suppressor Protein p53; D004247:DNA; D012697:Serine Endopeptidases; D003546:Cysteine Endopeptidases; D016333:HIV Protease; D002166:Camptothecin