Specific Proteolytic Cleavage of Poly(ADP-ribose) Polymerase: An Early Marker of Chemotherapy-induced Apoptosis

Scott H. Kaufmann, Serge Desnoyers, Yvonne Ottaviano, Nancy E. Davidson, Guy G. Poirier

Research output: Contribution to journalArticle

1619 Scopus citations

Abstract

Apoptosis is a morphologically and biochemically distinct form of cell death that occurs under a variety of physiological and pathological conditions. In the present study, the proteolytic cleavage of poly(ADP-ribose) polymerase (pADPRp) during the course of chemotherapy-induced apoptosis was examined. Treatment of HL-60 human leukemia cells with the topoisomerase II-directed anticancer agent etoposide resulted in morphological changes characteristic of apoptosis. Endonucleolytic degradation of DNA to generate nucleosomal fragments occurred simultaneously. Western blotting with epitope-specific monoclonal and polyclonal antibodies revealed that these characteristic apoptotic changes were accompanied by early, quantitative cleavage of the Mr 116,000 pADPRp polypeptide to an Mr ~25,000 fragment containing the amino-terminal DNA-binding domain of pADPRp and an Mr ~85,000 fragment containing the automod-ification and catalytic domains. Activity blotting revealed that the Mr ~85,000 fragment retained basal pADPRp activity but was not activated by exogenous nicked DNA. Similar cleavage of pADPRp was observed after exposure of HL-60 cells to a variety of chemotherapeutic agents including cis-diaminedichloroplatinum(II), colcemid, 1-β-D-arabinofura-nosylcytosine, and methotrexate; to γ-irradiation; or to the protein synthesis inhibitors puromycin or cycloheximide. Similar changes were observed in MDA-MB-468 human breast cancer cells treated with trifluorothymidine or 5-fluoro-2’-deoxyuridine and in γ-irradiated or glu-cocorticoid-treated rat thymocytes undergoing apoptosis. Treatment with several compounds (tosyl-L-lysine chloromethyl ketone, tosyl-L-phenylala-nine chloromethyl ketone, JV-ethylmaleimide, iodoacetamide) prevented both the proteolytic cleavage of pADPRp and the internucleosomal fragmentation of DNA The results suggest that proteolytic cleavage of pADPRp, in addition to being an early marker of chemotherapy-induced apoptosis, might reflect more widespread proteolysis that is a critical biochemical event early during the process of physiological cell death.

Original languageEnglish (US)
Pages (from-to)3976-3985
Number of pages10
JournalCancer research
Volume53
Issue number17
StatePublished - Sep 1993

ASJC Scopus subject areas

  • Oncology
  • Cancer Research

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    Kaufmann, S. H., Desnoyers, S., Ottaviano, Y., Davidson, N. E., & Poirier, G. G. (1993). Specific Proteolytic Cleavage of Poly(ADP-ribose) Polymerase: An Early Marker of Chemotherapy-induced Apoptosis. Cancer research, 53(17), 3976-3985.