Internucleosomal DNA cleavage in apoptotic WEHI 231 cells is mediated by a chymotrypsin-like protease.
Although several lines of evidence support a role for serine proteases in apoptosis, little is known about the mechanisms involved. In the present study, we have examined the apoptosis-inducing potential and dissected the death-signalling pathways of N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and N-tosyl-L-lysine chloromethyl ketone (TLCK), inhibitors of chymotrypsin- and trypsin-like proteases, respectively. Our results designate two distinct roles for serine proteases. Firstly, we show that both inhibitors induce biochemical and morphological characteristics of apoptosis, including proteolysis of poly(ADP-ribose) polymerase 1 (PARP-1) and inhibitor of caspase-activated DNase (ICAD), as well as mitochondrial dysfunction, and that their action is abrogated by the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp.fluoromethylketone (z-VAD.fmk). These results suggest that inhibition of anti-apoptotic serine proteases governs the onset of the caspase-dependant apoptotic cascade. Secondly, we also demonstrate the involvement of a serine protease in the terminal stage of apoptosis. We showed that chymotrypsin-like protease activity is required for internucleosomal DNA fragmentation in apoptotic cells. Hence, DNA fragmentation is abrogated in TPCK-pre-treated WEHI 231 cells undergoing apoptosis triggered either by anti-IgM or TLCK. These results indicate that internucleosomal DNA cleavage in apoptotic cells is mediated by a chymotrypsin-like protease.
Murn
Jernej
J
Faculty of Pharmacy, University of Ljubljana, Askerceva 7, 1000 Ljubljana, Slovenia.
Urleb
Uros
U
Mlinaric-Rascan
Irena
I
D017209:Apoptosis; D015153:Blotting, Western; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D004247:DNA / Q000378:metabolism*; D004789:Enzyme Activation; D005434:Flow Cytometry; D006868:Hydrolysis; D008564:Membrane Potentials; D010539:Permeability; D012697:Serine Endopeptidases / Q000378:metabolism*
D004247:DNA; D012697:Serine Endopeptidases; D020169:Caspases