title: Internucleosomal DNA cleavage triggered by plasma membrane damage during necrotic cell death. Involvement of serine but not cysteine proteases.
abstract: Autolytic DNA breakdown, detected as smears in electrophoretic gels, is a late event in necrosis. On the other hand, internucleosomal DNA cleavage, visualized as ladders, is thought to be a hallmark of apoptosis. We now report that this specific form of DNA fragmentation also occurs during necrosis and is an early event but appears to be triggered by proteolytic mechanisms significantly different from those documented in apoptosis. Treatment of MDCK cells with a mitochondrial uncoupler and a Ca2+ ionophore led to ATP depletion, necrotic morphology, and progressive fragmentation of DNA in an internucleosomal or ladder pattern. DNA breakdown was immediately preceded by increased permeability of the plasma membrane to macromolecules. Provision of glycine along with the noxious agents did not modify the extent of ATP depletion, but prevented plasma membrane damage. This was accompanied by complete inhibition of DNA fragmentation. Internucleosomal DNA cleavage was observed also during necrosis after rapid permeabilization of plasma membranes by detergents or streptolysin-O in hepatocytes, thymocytes, and P19, Jurkat, and MDCK cells. DNA fragmentation associated with necrosis was Ca2+/Mg2+ dependent, was suppressed by endonuclease inhibitors, and was abolished by serine protease inhibitors but not by inhibitors of interleukin-1 beta converting enzyme (ICE)-related proteases or caspases. Moreover, unlike apoptosis, it was not accompanied by caspase-mediated proteolysis. On the other hand, the cleavage-site-directed chymotryptic inhibitor N-tosyl-L-phenylalanyl-chloromethyl ketone (TPCK) suppressed 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 during necrosis not involving the participation of caspases. In addition, they indicate that techniques based on double-strand DNA breaks may not reliably differentiate between apoptosis and necrosis.
authors.affiliation: Department of Pathology, University of Texas Health Science Center at San Antonto 78284-7750, USA.
authors.forename: Z
authors.initials: Z
authors.lastname: Dong
authors.forename: P
authors.initials: P
authors.lastname: Saikumar
authors.forename: J M
authors.initials: JM
authors.lastname: Weinberg
authors.forename: M A
authors.initials: MA
authors.lastname: Venkatachalam
mesh_terms: D000255:Adenosine Triphosphate / Q000172:deficiency; D000818:Animals; D002118:Calcium / Q000502:physiology; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology* / Q000648:ultrastructure; D002463:Cell Membrane Permeability / Q000502:physiology; D003546:Cysteine Endopeptidases / Q000502:physiology*; D004247:DNA / Q000187:drug effects / Q000378:metabolism*; D004285:Dogs; D004720:Endonucleases / Q000037:antagonists & inhibitors; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008274:Magnesium / Q000502:physiology; D009336:Necrosis; D009707:Nucleosomes / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000502:physiology*
chemical_list: D004791:Enzyme Inhibitors; D009707:Nucleosomes; D000255:Adenosine Triphosphate; D004247:DNA; D004720:Endonucleases; D012697:Serine Endopeptidases; D003546:Cysteine Endopeptidases; D008274:Magnesium; D002118:Calcium