Role of proteolysis in apoptosis: involvement of serine proteases in internucleosomal DNA fragmentation in immature thymocytesl VALERIE M. WEAVER Apoptosis Research Group, Institute for Biological Sciences, National Research Council of Canada, Ottawa, ON KIA OR6, Canada BOLESLAW LACH Department of Laboratory Medicine (Neuropathology), University of Ottawa - Ottawa Civic Hospital, Ottawa, ON KI Y 4E9, Canada AND P. ROY WALKER AND MARIANNA SIKORSKA' Apoptosis Research Group, Institute for Biological Sciences, National Research Council of Canada, Ottawa, ON KIA OR6, Canada Received November 2, 1993 WEAVER, V.M., LACH, B., WALKER, P.R., and SIKORSKA, M. 1993. Role of proteolysis in apoptosis: involvement of serine proteases in internucleosomal DNA fragmentation in immature thymocytes. Biochem. Cell Biol. 71: 488-500. Three chemically distinct serine, but not cysteine, protease inhibitors (phenylmethylsulphonyl fluoride, N-tosyl-L- phenylalanylchloromethyl ketone and 34-dichloroisocoumarin) prevented, in a dose-dependent manner, the characteristic apoptotic internucleosomal DNA cleavage (DNA ladder) typically observed in thymoc es in response to dexamethasone f and teniposide VM-26. This effect was not the result of a direct inhibition of the Ca +,~g'+-dependent endonuclease, since oligonucleosomal DNA cleavage occurred in the presence of these inhibitors in isolated nuclei. The proteolytic step occurred at a very early stage of apoptosis, and preincubation of thymocytes with the inhibitors before dexamethasone or teniposide VM-26 were added irreversibly suppressed ladder formation. This implied that the cellular effector(s) of these compounds preexisted and were not resynthesized in response to the inducers of apoptosis. Serine protease inhibitors also suppressed apoptotic cell shrinkage and complete nuclear collapse, suggesting that these morphological changes were directly related to internucleosornal fragmentation of DNA. However, the serine protease inhibitors did not prevent high molecular weight DNA cleavage (> 50 kilobases) that preceded the ladder formation and thymocytes still died by apoptosis. This supported the view that internucleosomal DNA cleavage, considered to be the biochemical marker of apoptosis, might in fact be a late and dispensable step and that the newly described high molecular weight DNA cleavage might be a better indicator of apoptosis. Key words: serine protease, apoptosis, internucleosomal DNA fragmentation, high molecular weight DNA cleavage, protease inhibitors. WEAVER, V.M., LACH, B., WALKER, P.R., et SIKORSKA, M. 1993. Role of proteolysis in apoptosis: involvement of serine proteases in internucleosomal DNA fragmentation in immature thymocytes. Biochem. Cell Biol. 71 : 488-500. Le fluorure de phCnylmethylsulfonyle, la N-tosyl L-phenylalanylchlorom~thylcttone et la 3,4-dichloroisocoumarine sont trois inhibiteurs de la serine-protease, mais non de la cysteine-protease, de natures chimiques distinctes. Proportion- nellement a leur dose, ils empCchent la fragmentation de I'ADN internucleosomique (tchelle d'ADN), caracteristique de I'apoptose, qui est typiquement observte dans les thymocytes a la suite d'un traitement a la dexamkthasone et au teniposide VM-26. Cet effet ne resulte pas d'une inhibition directe des endonucleases ~ a ' + , ~ ~ ~ + - d e p e n d a n t e s puisque I'ADN oligonucl~osomique dans des noyaux isoles se fragmente en presence de ces inhibiteurs. L'etape de la proteolyse se produit A un stade t r b prtcoce de I'apoptose et la preincubation des thymocytes en presence de ces inhibiteurs avant l'exposition a la dkamethasone ou au tkniposide VM-26 empCche la formation de l'echelle de fagon irreversible. Cela indique que le ou les effecteurs cellulaires de ces inhibiteurs existent dejk et qu'ils ne sont pas synthttists de nouveau en rkponse aux inducteurs de I'apoptose. Les inhibiteurs de la serine-protease suppriment tgalement le rttrtcissement des cellules en apoptose et la contraction nucleaire totale, ce qui suggkre que ces changements morphologiques sont directement lies a la fragmentation de I'ADN internucleosomique. Cependant, les inhibiteurs de la shine-protease n'empCchent pas la fragmentation de 1'ADN de masse moltculaire elevee (> 50 kilobases) precedant la formation de l'echelle et les thymocytes meurent neanmoins par apoptose. Ceci appuie le concept que la fragmentation de I'ADN internucltosomique, considtree comme le signe biochimique de l'apoptose, en fait, pourrait Ctre une Ctape tardive et non indispensable et que la fragmentation de 1'ADN de masse moleculaire elevke nouvellement dkcrite pourrait Ctre un meilleur indicateur de I'apoptose. Mots clPs : serine-protease, apoptose, fragmentation de I'ADN internuclCosomique, fragmentation de I'ADN de masse molkculaire elevte, inhibiteurs des protkases. [Traduit par la redaction] ABBREVIATIONS: Dex, dexamethasone; VM-26, teniposide VM-26; kb, kilobase(s); CTL, cytotoxic T lymphocyte; DCI, 3,4-dichloroisocoumarin; DMSO, dimethyl sulfoxide; PMSF, phenylmethylsulfonyl fluoride; TPCK, N-tosyl-L-phenylalanylchloromethyl ketone; PBS, phosphate-buffered saline; DTT, dithiothreitol; SDS, sodium dodecyl sulfate; CAGE, conventional agarose gel electro- phoresis; bp, base pair(s); PFGE, pulsed field gel electrophoresis; Leup, leupeptin; Cal-Inh, calpain I1 inhibitor; NK, natural killer; NuMa, nuclear mitotic apparatus protein. 'NRCC 37371. '~uthor to whom all correspondence should be addressed. Printed in Canada / Imprime au Canada Biochem. Cell Biol. Downloaded from www.nrcresearchpress.com by San Francisco (UCSF) on 11/24/14 For personal use only.