Specific Proteolytic Cleavage of Poly(ADP-ribose) Polymerase: An Early Marker of Chemotherapy-induced Apoptosis1

Apoptosis ¡sa morphologically and biochemically distinct form of cell death that occurs under a variety of physiological and pathological con ditions. 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 H-directed anticancer agent etoposide resulted in morpho logical changes characteristic of apoptosis. Endonucleolytic degradation of DNA to generate nucleosomal fragments occurred simultaneously. West ern blotting with epitope-specific monoclonal and polyclonal antibodies revealed that these characteristic apoptotic changes were accompanied by early, quantitative cleavage of the U, 116,000 pADPRp polypeptide to an U, —¿ 25,0110 fragment containing the amino-terminal DNA-binding do main of pADPRp and an \t, —¿ 85,000 fragment containing the automod ification and catalytic domains. Activity blotting revealed that the U, —¿ 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 m-diaminedichloroplatinum(II), colcemid, 1-ß-n-arabinofuranosylcytosine, and methotrexate; to y-irradiation; or to the protein syn thesis inhibitors puromycin or cycloheximide. Similar changes were ob served in MDA-MB-468 human breast cancer cells treated with trifluorothymidine or 5-fluoro-2'-deoxyuridine and in y-irradiated or glucocorticoid-treated rat thymocytes undergoing apoptosis. Treatment with several compounds (tosyl-i.-lysine chloromethyl ketone, tosyl-i.-phenylalanine chloromethyl ketone, /V-ethylmaleimide, iodoacetamide) prevented both the proteolytic cleavage of pADPRp and the internucleosomal frag mentation 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.