Kaufmann S H, Desnoyers S, Ottaviano Y, Davidson N E, Poirier G G
Oncology Center, Johns Hopkins Hospital, Baltimore, Maryland 21287.
Cancer Res. 1993 Sep 1;53(17):3976-85.
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) approximately 25,000 fragment containing the amino-terminal DNA-binding domain of pADPRp and an M(r) approximately 85,000 fragment containing the automodification and catalytic domains. Activity blotting revealed that the M(r) approximately 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-beta-D-arabinofuranosylcytosine, and methotrexate; to gamma-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 gamma-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.
细胞凋亡是一种在形态和生化方面都有明显特征的细胞死亡形式，它发生在多种生理和病理条件下。在本研究中，我们检测了化疗诱导的细胞凋亡过程中聚（ADP - 核糖）聚合酶（pADPRp）的蛋白水解切割情况。用拓扑异构酶II导向的抗癌药物依托泊苷处理HL - 60人白血病细胞，导致出现细胞凋亡的形态学变化。同时发生了DNA的内切核酸酶降解，产生核小体片段。用表位特异性单克隆和多克隆抗体进行蛋白质印迹分析表明，这些特征性的凋亡变化伴随着M(r) 116,000的pADPRp多肽早期的定量切割，形成一个M(r)约为25,000的片段，该片段包含pADPRp的氨基末端DNA结合结构域，以及一个M(r)约为85,000的片段，该片段包含自身修饰和催化结构域。活性印迹分析表明，M(r)约为85,000的片段保留了基础水平的pADPRp活性，但不能被外源性切口DNA激活。在HL - 60细胞暴露于多种化疗药物（包括顺二氯二氨铂(II)、秋水仙酰胺、1 - β - D - 阿拉伯呋喃糖基胞嘧啶和甲氨蝶呤）后，以及在接受γ射线照射后，或者在接触蛋白质合成抑制剂嘌呤霉素或环己酰亚胺后，均观察到pADPRp的类似切割。在用三氟胸苷或5 - 氟 - 2'-脱氧尿苷处理的MDA - MB - 468人乳腺癌细胞以及在接受γ射线照射或糖皮质激素处理后发生凋亡的大鼠胸腺细胞中也观察到了类似变化。用几种化合物（甲苯磺酰 - L - 赖氨酸氯甲基酮、甲苯磺酰 - L - 苯丙氨酸氯甲基酮、N - 乙基马来酰亚胺、碘乙酰胺）处理可同时阻止pADPRp的蛋白水解切割和DNA的核小体间断裂。结果表明，pADPRp的蛋白水解切割除了是化疗诱导细胞凋亡的早期标志物外，可能还反映了更广泛的蛋白水解过程，这是生理细胞死亡过程早期的一个关键生化事件。
