INTERNUCLEOSOMAL DNA CLEAVAGE TRIGGERED BY PLASMA-MEMBRANE DAMAGE DURING NECROTIC CELL-DEATH - INVOLVEMENT OF SERINE BUT NOT CYSTEINE PROTEASES

Autolytic DNA breakdown, detected as smears in electrophoretic gels, i s a late event in necrosis. On the other hand, internucleosomal DNA cl eavage, visualized as ladders, is thought to be a hallmark of apoptosi s. We now report that this specific form of DNA fragmentation also occ urs during necrosis and is an early event but appears to be triggered by proteolytic mechanisms significantly different from those documente d in apoptosis. Treatment of MDCK cells with a mitochondrial uncoupler and a Ca2+ ionophore led to ATP depletion, necrotic morphology, and p rogressive fragmentation of DNA in an internucleosomal or ladder patte rn. DNA breakdown was immediately preceded by increased permeability o f the plasma mem brane to macromolecules. Provision of glycine along w ith the noxious agents did not modify the extent of ATP depletion, but prevented plasma membrane damage. This was accompanied by complete in hibition of DNA fragmentation. Internucleosomal DNA cleavage was obser ved also during necrosis after rapid permeabilization of plasma membra nes by detergents or streptolysin-O in hepatocytes, thymocytes, and P1 9, Jurkat, and MDCK cells. DNA fragmentation associated with necrosis was Ca2+/Mg2+ dependent, was suppressed by endonuclease inhibitors, an d was abolished by serine protease inhibitors but not by inhibitors of interleukin-1 beta converting enzyme (ICE)-related proteases or caspa ses. Moreover, unlike apoptosis, it was not accompanied by caspase-med iated proteolysis. On the other hand, the cleavage-site-directed chymo tryptic inhibitor N-tosyl-L-phenylalanyl-chloromethyl ketone (TPCK) su ppressed DNA fragmentation not only in necrotic cells but also during Fas-mediated apoptosis, without inhibiting caspase-related proteolysis . The results suggest a novel pathway of endonuclease activation durin g necrosis not involving the participation of caspases. In addition, t hey indicate that techniques based on double-strand DNA breaks may not reliably differentiate between apoptosis and necrosis.