SPECIFIC PROTEOLYTIC CLEAVAGE OF POLY(ADP-RIBOSE) POLYMERASE - AN EARLY MARKER OF CHEMOTHERAPY-INDUCED APOPTOSIS

Apoptosis is a morphologically and biochemically distinct form of cell death that occurs under a variety of physiological and pathological c onditions. 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 t he topoisomerase II-directed anticancer agent etoposide resulted in mo rphological changes characteristic of apoptosis. Endonucleolytic degra dation of DNA to generate nucleosomal fragments occurred simultaneousl y. Western blotting with epitope-specific monoclonal and polyclonal an tibodies revealed that these characteristic apoptotic changes were acc ompanied by early, quantitative cleavage of the M(r) 116,000 pADPRp po lypeptide to an M(r) approximately 25,000 fragment containing the amin o-terminal DNA-binding domain of pADPRp and an M(r) approximately 85,0 00 fragment containing the automodification and catalytic domains. Act ivity blotting revealed that the M(r) approximately 85,000 fragment re tained 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-diaminedic hloroplatinum(II), colcemid, 1-beta-D-arabinofuranosylcytosine, and me thotrexate; to gamma-irradiation; or to the protein synthesis inhibito rs puromycin or cycloheximide. Similar changes were observed in MDA-MB -468 human breast cancer cells treated with trifluorothymidine or 5-fl uoro-2'-deoxyuridine and in gamma-irradiated or glucocorticoid-treated rat thymocytes undergoing apoptosis. Treatment with several compounds (tosyl-L-lysine chloromethyl ketone, tosyl-L-phenylalanine chlorometh yl ketone, N-ethylmaleimide, iodoacetamide) prevented both the proteol ytic 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 refl ect more widespread proteolysis that is a critical biochemical event e arly during the process of physiological cell death.