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

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 M_r 116,000 pADPRp polypeptide to an M_r ∼25,000 fragment containing the amino-terminal DNA-binding domain of pADPRp and an M_r ∼85,000 fragment containing the automodification and catalytic domains. Activity blotting revealed that the M_r ∼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-arabinofuranosylcytosine, 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 glucocorticoid-treated rat thymocytes undergoing apoptosis. Treatment with several compounds (tosyl-l-lysine chloromethyl ketone, tosyl-l-phenylalanine chloromethyl ketone, N-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.