Internucleosomal DNA cleavage triggered by plasma membrane damage during necrotic cell death: Involvement of serine but not cysteine proteases

Zheng Dong, Pothana Saikumar, Joel M. Weinberg, Manjeri A. Venkatachalam

Research output: Contribution to journalArticle

207 Citations (Scopus)

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- 1β 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.

Original languageEnglish (US)
Pages (from-to)1205-1213
Number of pages9
JournalAmerican Journal of Pathology
Volume151
Issue number5
StatePublished - Nov 1 1997

Fingerprint

DNA Cleavage
Cysteine Proteases
Serine
DNA Fragmentation
Cell Death
Necrosis
Cell Membrane
Caspases
Apoptosis
Madin Darby Canine Kidney Cells
Endonucleases
Proteolysis
Adenosine Triphosphate
Caspase 1
Serine Proteinase Inhibitors
Jurkat Cells
Double-Stranded DNA Breaks
DNA
Ionophores
Thymocytes

ASJC Scopus subject areas

  • Pathology and Forensic Medicine

Cite this

Internucleosomal DNA cleavage triggered by plasma membrane damage during necrotic cell death : Involvement of serine but not cysteine proteases. / Dong, Zheng; Saikumar, Pothana; Weinberg, Joel M.; Venkatachalam, Manjeri A.

In: American Journal of Pathology, Vol. 151, No. 5, 01.11.1997, p. 1205-1213.

Research output: Contribution to journalArticle

@article{84c4b449e55b4dbfb38b7f1b95dc1270,
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- 1β 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.",
author = "Zheng Dong and Pothana Saikumar and Weinberg, {Joel M.} and Venkatachalam, {Manjeri A.}",
year = "1997",
month = "11",
day = "1",
language = "English (US)",
volume = "151",
pages = "1205--1213",
journal = "American Journal of Pathology",
issn = "0002-9440",
publisher = "Elsevier Inc.",
number = "5",

}

TY - JOUR

T1 - Internucleosomal DNA cleavage triggered by plasma membrane damage during necrotic cell death

T2 - Involvement of serine but not cysteine proteases

AU - Dong, Zheng

AU - Saikumar, Pothana

AU - Weinberg, Joel M.

AU - Venkatachalam, Manjeri A.

PY - 1997/11/1

Y1 - 1997/11/1

N2 - 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- 1β 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.

AB - 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- 1β 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.

UR - http://www.scopus.com/inward/record.url?scp=0030663451&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0030663451&partnerID=8YFLogxK

M3 - Article

C2 - 9358745

AN - SCOPUS:0030663451

VL - 151

SP - 1205

EP - 1213

JO - American Journal of Pathology

JF - American Journal of Pathology

SN - 0002-9440

IS - 5

ER -